WO2024047185A1 - Mass spectroscopy assay for detecting o-beta-linked n-acetylglucosaminylated tau peptides - Google Patents

Mass spectroscopy assay for detecting o-beta-linked n-acetylglucosaminylated tau peptides Download PDF

Info

Publication number
WO2024047185A1
WO2024047185A1 PCT/EP2023/073945 EP2023073945W WO2024047185A1 WO 2024047185 A1 WO2024047185 A1 WO 2024047185A1 EP 2023073945 W EP2023073945 W EP 2023073945W WO 2024047185 A1 WO2024047185 A1 WO 2024047185A1
Authority
WO
WIPO (PCT)
Prior art keywords
residue
tau
seq
peptides
glcnac
Prior art date
Application number
PCT/EP2023/073945
Other languages
French (fr)
Inventor
Sebastiaan BIJTTEBIER
Alexis BRETTEVILLE
Andreas Ebneth
Clara THEUNIS
Lieve DILLEN
Dina RODRIGUES MARTINS
Original Assignee
Janssen Pharmaceutica Nv
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Janssen Pharmaceutica Nv filed Critical Janssen Pharmaceutica Nv
Publication of WO2024047185A1 publication Critical patent/WO2024047185A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • sequence listing of the present application is submitted electronically via The United States Patent and Trademark Center Patent Center as an XML formatted sequence listing with a file name “JAB7172WOPCTl_SEQLIST.xml”, creation date of August 29, 2023, and a size of 43 kilobytes (KB).
  • This sequence listing submitted is part of the specification and is herein incorporated by reference in its entirety.
  • the present invention relates to methods for detecting neurodegeneration.
  • the invention relates to methods of measuring an amount of O-0-linked N-acetylglucosaminylated tau (O-GlcNAc-tau) protein species in a biologic sample and uses thereof.
  • O-GlcNAc-tau O-0-linked N-acetylglucosaminylated tau
  • AD Alzheimer’s Disease
  • AD is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death.
  • AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States.
  • AD has been observed in ethnic groups worldwide and presents a major present and future public health problem.
  • the brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles.
  • senile or amyloid
  • amyloid angiopathy amyloid deposits in blood vessels
  • neurofibrillary tangles Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD.
  • Neurofibrillary tangles are primarily composed of aggregates of hyper-phosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization.
  • tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the primary function of tau.
  • Tau that is bound to the axonal microtubule lattice is in a hypo-phosphorylation state, while aggregated tau in AD is hyper-phosphorylated, providing unique epitopes that are distinct from the physiologically active pool of tau (Iqbal et al., Curr Alzheimer Res. 7(8): 656-664, 2010).
  • O-GlcNAcylation O-0-linked N-acetylglucosaminylation (O-GlcNAcylation), regulated by two antagonist enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), modulates tau phosphorylation and slows down its aggregation in vitro (Cantrelle et al., Frontiers in Molecular Neuroscience, 14: 661368, 2021). Therefore, the level of OGA modulation may be indirectly related to the process involving the (9-GlcN Ac-mediated regulation of enzymes implicated in phosphorylation dynamics or other actors in tau pathology (Cantrelle et al., 2021).
  • tau species found in CSF differ from tau in brain: the predominant forms of tau (99.9%) in CSF are C-terminally truncated containing the mid-domain but lacking the microtubule binding region and C-terminus, with cleavage between amino acid (AA) residues 222 and 225 (based on tau isoform 2N4R)(Sato, 2018).
  • One exemplary embodiment of the present invention is directed to a method for detecting OGA inhibition in a brain of a subject.
  • An OGA inhibitor may have been administered to the subject.
  • the method comprises step a) detecting O-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample (e.g., cerebrospinal fluid (CSF)) obtained from the subject.
  • CSF cerebrospinal fluid
  • OGA inhibition in the brain of the subject is determined to be present when the amount of O- GlcNAcylated tau peptides is above a predetermined threshold value.
  • the tau protein in the biologic sample is concentrated by immunoprecipitation before the detecting of O- GlcNAcylation.
  • step a) comprises detecting (9-GlcN Acylation in (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample obtained from the subject.
  • step a) comprises detecting O-GlcNAcylation in at least one of residue 184, 185, 191, and 208 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample obtained from the subject.
  • step a) comprises detecting O- GlcNAcylation in (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 of the tau protein to determine an amount of O-GlcNAcylated tau peptides in a biologic sample obtained from the subject.
  • the O-GlcNAcylation is detected by liquid chromatography mass spectrometry (LC-MS), such as nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS) or ultra-high performance liquid chromatography- MS/MS (UHPLC- MSMS).
  • LC-MS liquid chromatography mass spectrometry
  • nLC-HRMS nanoflow liquid chromatography - high resolution mass spectrometry
  • UHPLC- MSMS ultra-high performance liquid chromatography- MS/MS
  • the (9-GlcN Acylation is detected by an immunoassay.
  • the immunoassay is an ultrasensitive assay, such as a single-molecule array (SIMOA) assay.
  • SIMOA single-molecule array
  • An assay method of detecting O-GlcNAc-tau peptides comprises obtaining a biologic sample from a human subject, and contacting the biologic sample (e.g., CSF) with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support (e.g., magnetic beads) to isolate tau protein from the biologic sample.
  • a biologic sample e.g., CSF
  • an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support (e.g., magnetic beads) to isolate tau protein from the biologic sample.
  • the method further comprises digesting the isolated tau protein with at least one enzyme, and detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry (e.g., nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS)) to determine an amount of O-GlcNAcylated tau peptides in the biologic sample.
  • liquid chromatography mass spectrometry e.g., nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS)
  • nLC-HRMS nanoflow liquid chromatography - high resolution mass spectrometry
  • residue 400 are detected.
  • the immunoprecipitation antibody used in the assay method binds to an epitope between amino acids 163 to 174 of human tau protein or an epitope between amino acids 219 to 226 of the human tau protein.
  • the immunoprecipitation antibody comprises an immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 comprising the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively and an immunoglobulin light chain LCDR1, LCDR2 and LCDR3 comprising the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively.
  • the immunoprecipitation antibody comprises a variable heavy chain region (VH) comprising the polypeptide sequence of SEQ ID NO: 8 and a variable light chain region (VL) comprising the polypeptide sequence of SEQ ID NO: 9.
  • the assay method digests the isolated tau protein with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested peptides.
  • the isolated tau protein is divided into at least five aliquots and digested with the at least one enzyme, wherein the aliquots comprise: (1) an aliquot digested with trypsin; (2) an aliquot digested with asp-N; (3) an aliquot digested with glu-C; (4) an aliquot digested with trypsin followed by asp-N; and (5) an aliquot digested with trypsin followed by glu-C.
  • the LC-MS (e.g. , nLC-HRMS) may be used to detect O-GlcN Acylation at residue 184 or 185 by detecting presence of a polypeptide having a sequence of SEQ ID NO: 16 or 17.
  • ( -GlcN Acylation may be detected at residue 184 or 185 is detected by detecting ion transition from 600.3 m/z to 1001.5 m/z, 600.3 m/z to 996.5 m/z, or from 600.3 m/z to 798.4 m/z.
  • O-GlcN Acylation at residue 191 may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 18.
  • (9-GlcN Acylation at residue 191 may be detected by detecting ion transition from 538.9 m/z to 959.5 m/z, or 538.9 m/z to 756.4 m/z.
  • O-GlcNAcylation may be detected at residue 208 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 19.
  • (9-GlcN Acylation at (ii) residue 208 is detected by detecting ion transition from 798.9 m/z to 619.3 m/z, 798.9 m/z to 874.4 m/z, or from 798.9 m/z to 1115.5 m/z.
  • O-GlcN Acylation may be detected at residue 400 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 20.
  • O-GlcNAcylation at residue 400 may be detected by detecting ion transition from 652.8 m/z to 1101.4 m/z.
  • another assay method of detecting O- GlcNAc-tau peptides comprises obtaining a biologic sample (c.g, CSF) from a human subject and contacting the biologic sample with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support to isolate tau protein from the biologic sample.
  • a biologic sample c.g, CSF
  • an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support to isolate tau protein from the biologic sample.
  • the method also comprises labelling O-GlcNAc sites of the isolated tau protein with biotin to produce biotinylated O-GlcNAc peptides, digesting the biotinylated O-GlcNAc peptides with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested biotinylated peptides, and immobilizing the digested biotinylated peptides to a solid support ( .g, streptavidin beads).
  • a solid support .g, streptavidin beads
  • the method further comprises reacting the digested biotinylated peptides with Na2SCh in a P-elimination - Michael addition reaction to release sulfited peptides from the solid support.
  • the method further comprises detecting sulfite modification in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry to determine an amount of G-GlcN Acylated tau peptides in the biologic sample.
  • Fig. 1 shows a method for detecting O-GlcNAc-tau peptides according to an exemplary embodiment of the present application.
  • FIG. 2 shows extracted ion chromatograms of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 3 shows MS fragmentation spectra of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 4 shows extracted ion chromatograms of peptides HLSNVSSTGSI (SEQ ID NO: 36) and HLSNVSSTGSI*O-GlcNAc isomers generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 5 shows MS fragmentation spectra of peptides HLSNVSSTGSI (SEQ ID NO: 36) and HLSNVSSTGSI*O-GlcNAc isomers generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 6 shows extracted ion chromatograms of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 15) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 7 shows MS fragmentation spectra of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 15) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 8 shows extracted ion chromatograms of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • Fig. 9 shows MS fragmentation spectra of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • Fig. 9 shows MS fragmentation spectra of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • TPPSSGEPPK SEQ ID NO: 12
  • TPPSSGEPPK*O-GlcNAc SEQ ID NO: 16 or 17
  • FIG. 11 shows MS fragmentation spectra of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 12 shows extracted ion chromatograms of peptides QAAAQPHTEIPEGTTAE EAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGI GDTPSLEDEAAGHVTQAR*O-GlcNAc generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 13 shows MS fragmentation spectra of peptides QAAAQPTHEIPEGTTAEEAGI GDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGDTPSL EDEAAGHVT QAR*O-GlcNAc generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
  • FIG. 14 another method for detecting O-GlcNAc-tau peptides according to an exemplary embodiment of the present application described in Example 3.
  • Fig. 15 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), SPW- (O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR in the extract of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau generated according to an exemplary embodiment of the present application described in Example 3.
  • Fig. 16 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), SPW- (O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR in the depleted fraction after capture of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau peptides with streptavidin beads generated according to an exemplary embodiment of the present application described in Example 3. [0032] Fig.
  • FIG. 18 MS fragmentation spectrum of SPW-(SO 3 )S-GDTSPR (SEQ ID NO: 25) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
  • FIG. 19 shows MS fragmentation spectrum of SGYSSPGSPGTPG-(SO 3 )S-R (SEQ ID NO: 24) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
  • FIG. 20 shows MS fragmentation spectrum of TPPSSGEPPK-(SO 3 )S-GDR (SEQ ID NO: 23) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
  • Fig. 21A shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of Thiamet-G treated mice according to an exemplary embodiment of the present application described in Example 4.
  • Fig. 21B shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of Thiamet-G treated mice according to an exemplary embodiment of the present application described in Example 4.
  • Fig. 21C shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 4.
  • SI 84 or SI 85 SEQ ID NO: 16 or 17
  • FIG. 22A shows nLC-MSMS chromatograms of SPVV-((O-GlcNAc)S)-GDTSPR (SEQ ID NO: 20) and SPVVSGDTSPR (SEQ ID NO: 15) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
  • Fig. 22B shows nLC-MSMS chromatograms of SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) and SGYSSPGSPGTPGSR (SEQ ID NO: 14) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
  • Fig. 22C shows nLC-MSMS chromatograms of TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) and TPPSSGEPPKSGDR (SEQ ID NO: 13) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
  • Fig. 22D shows nLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) and TPPSSGEPPK (SEQ ID NO: 12) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • antibody refers to a specific protein capable of binding an antigen or portion thereof. These terms are used herein in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies (including murine, human, human-adapted, humanized and chimeric monoclonal antibodies) and antibody fragments.
  • antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen.
  • Antibody structures are well known.
  • Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • the antibodies of the present application can be of any of the five major classes or corresponding sub-classes.
  • the antibodies of the present application are IgGl , IgG2, IgG3 or IgG4.
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the present application can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the present application include heavy and/or light chain constant regions from mouse antibodies or human antibodies.
  • antibodies In addition to the heavy and light constant domains, antibodies contain light and heavy chain variable regions.
  • An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by “antigen-binding sites.”
  • the antigen-binding sites are defined using various terms and numbering schemes as follows:
  • the antigen-binding site has three hypervariable regions in each VH (Hl, H2, H3) and VL (LI, L2, L3). Numbering systems as well as annotation of CDRs and HVRs have been revised by Abhinandan and Martin (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008);
  • IMGT Another definition of the regions that form the antigen-binding site has been proposed by Lefranc (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003) based on the comparison of V domains from immunoglobulins and T-cell receptors.
  • the International ImMunoGeneTics (IMGT) database http:_//www_imgt_org) provides a standardized numbering and definition of these regions. The correspondence between CDRs, HVRs and IMGT delineations is described in Lefranc et al., 2003, Id.,-
  • the antigen-binding site can also be delineated based on “Specificity Determining Residue Usage” (SDRU) (Almagro, Mol Recognit. 17: 132-43, 2004), where SDR, refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact.
  • SDRU Specificity Determining Residue Usage
  • Framework or “framework sequence” is the remaining sequences within the variable region of an antibody other than those defined to be antigen-binding site sequences. Because the exact definition of an antigen-binding site can be determined by various delineations as described above, the exact framework sequence depends on the definition of the antigen-binding site.
  • the framework regions (FRs) are the more highly conserved portions of variable domains.
  • the variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively) which generally adopt a beta-sheet configuration, connected by the three hypervariable loops.
  • the hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817, 1992; Tramontane et al., J. Mol. Biol. 215:175-182, 1990).
  • the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a bispecific or multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antibody fragment such as, for example, a diabody, a Fab
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.
  • the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the constant region of the heavy chain.
  • the antigen-binding fragment comprises Fab and F(ab’).
  • epitope refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof, specifically binds.
  • Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • tau or “tau protein” refers to an abundant central and peripheral nervous system protein having multiple isoforms.
  • CNS human central nervous system
  • the isoforms differ from each other by the regulated inclusion of 0-2 N-terminal inserts, and 3 or 4 tandemly arranged microtubule-binding repeats, and are referred to as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R.
  • control tau refers to the tau 2N4R isoform of SEQ ID NO: 1 that is devoid of phosphorylation and other post-translational modifications.
  • tau includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full-length wild type tau.
  • the term “tau” also encompasses post- translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, ( -GlcN Acylation or phosphorylation.
  • O-GlcNAcylated tau peptides As used herein, the term “O-GlcNAcylated tau peptides,” “O-GlcNAcylated tau,” or “O-GlcNAcylated tau protein” means human tau protein or tau fragment that is GlcNAcylated at one or more residue of tau protein.
  • S184/S185 O-GIcN Acylated tau peptide means a human tau protein or tau fragment that is GlcNAcylated at residue 184 (SI 84) or residue 185 (SI 85) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S 191 O-GIcN Acylated tau peptide means a human tau protein or tau fragment that is GlcNAcylated at residue 191 (SI 91) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S208 O-GlcNAcylated tau peptide means a human tau protein or tau fragment that is GlcNAcylated at residue 208 (S208) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S400 O-GlcNAcylated tau peptide As used herein, the term “S400 O-GlcNAcylated tau peptide,” “S400 O-GlcNAcylated tau,” or “S400 O-GIcN Acylated tau protein” means a human tau protein or tau fragment that is GlcNAcylated at residue 400 (S400) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S 184/S185 sulfite modified tau peptide means a human tau protein or tau fragment that is sulfite modified at residue 184 (SI 84) or residue 185 (SI 85) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • SI 91 sulfite modified tau peptide means a human tau protein or tau fragment that is sulfite modified at residue 191 (SI 91) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S208 sulfite modified tau peptide means a human tau protein or tau fragment that is sulfite modified at residue 208 (S208) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • S400 sulfite modified tau peptide means a human tau protein or tau fragment that is sulfite modified at residue 400 (S400) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • immunoprecipitation antibody refers to an antibody that binds to an antigen of interest and is or becomes directly or indirectly linked to a solid support.
  • solid supports include, but are not limited to, microparticles or beads, such as magnetic beads, paramagnetic beads, agarose beads, or streptavidin beads.
  • immunoprecipitation antibodies include, but are not limited to, a monoclonal antibody that binds to tau.
  • the immunoprecipitation antibody does not bind to the N-terminal or the mid domain regions of the tau protein. More particularly, the immunoprecipitation antibody binds to tau in the proline rich region of the tau protein.
  • the immunoprecipitation antibody can be a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and/or amino acids 219 and 226 of tau protein, and the numbering of the positions is according to the numbering in SEQ ID NO: 1.
  • the immunoprecipitation antibody can be a monoclonal antibody comprising immunoglobulin heavy chain HCDR1 , HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7.
  • the immunoprecipitation antibody is PT9.
  • PT9 refers to an antibody that binds to tau an epitope between amino acids 163 and 174 and an epitope between amino acids 219 and 226 of tau protein, has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 8, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 9. More specifically, PT9 antibody has an immunoglobulin heavy chain comprising the polypeptide sequence of SEQ ID NO: 10 and an immunoglobulin light chain comprises the polypeptide sequence of SEQ ID NO: 11.
  • the term “subject” refers to an animal, and preferably a mammal.
  • the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, mouse, rat, rabbit, guinea pig, marmoset or mouse) or a primate (e.g., a monkey, chimpanzee, or human).
  • a non-primate e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, mouse, rat, rabbit, guinea pig, marmoset or mouse
  • a primate e.g., a monkey, chimpanzee, or human.
  • the subject is a human.
  • tauopathy encompasses any neurodegenerative disease that involves the pathological aggregation of tau within the brain.
  • other exemplary tauopathies are frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corti cobasal degeneration, Pick’s disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myot
  • FTDP-17 frontotemporal dementia with parkinsonism linked to chromosome
  • amyloidogenic disease includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils.
  • exemplary amyloidogenic diseases include, but are not limited to systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson’s disease, Huntington’s disease, fronto-temporal dementia, and the prion- related transmissible spongiform encephalopathies (kuru and Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep and cattle, respectively).
  • Different amyloidogenic diseases are defined or characterized by the nature of the polypeptide component of the fibrils deposited.
  • P-amyloid protein e.g., wild-type, variant, or truncated P-amyloid protein
  • A0 a “disease characterized by deposits of A0” or a “disease associated with deposits of A0”, e.g., in the brain of a subject or patient.
  • the terms “0-amyloid protein,” “[3-amyloid peptide,” “P-amyloid,” “AP” and “Ap peptide” are used interchangeably herein.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.
  • the terms “increase” and “decrease” refer to differences in the quantity of a particular biomarker in a sample as compared to a control or reference level.
  • the quantity of particular peptide may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level.
  • an “increase of a level” or “decrease of a level” may be a difference between the level of biomarker present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more.
  • an “increase of a level” or “decrease of a level” may be a statistically significant difference between the level of the biomarker present in a sample as compared to a control.
  • a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
  • the reference or control can be, for example, a sample from a healthy individual, or a sample taken from the same individual at an earlier time point, such as a time point prior to administration of a therapeutic or an earlier time point during a therapeutic regimen.
  • isolated means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • isolated nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • nucleic acid molecule refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • moduleating, ameliorating, or treating includes prophylaxis of a physical and/or mental condition or amelioration or elimination of the developed physical and/or mental condition once it has been established or alleviation of the characteristic symptoms of such condition.
  • the present application provides assays and methods for detecting singly- or multiply - ( -GlcN Acylated tau peptides in a biologic sample.
  • the sample used in assays and methods of the present application may be a brain homogenate or a cerebrospinal fluid (CSF) sample.
  • the biologic sample can be from a mammal, such as a mouse, non-human primate, or human.
  • the biologic sample is a CSF sample from a human.
  • the assays and methods of the present application are directed to measurement of O-GlcNAcylated tau peptides in biologic samples by detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in the biologic sample.
  • the assays and methods of the present application detect O-GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208 or any combination thereof are detected.
  • the assays and methods detect (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208.
  • the assays and methods of the present application detect (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, (iv) residue 400 or any combination are detected.
  • the assays and methods detect O- GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400.
  • the amount of O-GlcNAcylated tau peptides may be quantified for use in various diagnostic purposes, monitoring the level of O-GlcNAc hydrolase (OGA) inhibition, monitoring activity of an O-GlcNAc hydrolase (OGA) inhibitor in a subject, monitoring the effectiveness of a treatment such as an OGA inhibitor, identifying a subject suitable for treatment with an O-GlcNAc hydrolase inhibitor, identifying a subject suitable for a treatment for tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), pre-screening subjects for PET imaging or other diagnostic tests for detection tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), identification of subjects for enrollment in clinical trials for an O-GlcNAc hydrolase inhibitor, identification of subjects for enrollment in clinical trials relating to tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), etc.
  • OGA O-GlcNAc hydrolase
  • OAA O-GlcNAc hydrolase
  • the assays and methods of the present application preferably include a step for isolating and/or concentration the tau protein from the biologic sample (e.g., human CSF) by immunoprecipitation before analysis of the sample to detect any O- GlcNAcylation.
  • the tau protein may be immunoprecipitated using an immunoprecipitation antibody which binds to tau.
  • the immunoprecipitation antibody selectively binds to human tau protein and is attached to or becomes attached to a solid phase during the process so that the immunoprecipitation antibody immobilizes tau protein in the sample to the solid phase.
  • the solid phase may be magnetic beads, paramagnetic beads, agarose beads, or streptavidin beads. Preferably, the solid phase is magnetic beads. Supernatant from the sample may be removed and thereby isolating and/or concentrating tau protein in the remainder of the sample.
  • the immunoprecipitation antibody is a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and/or amino acids 219 and 226 of tau protein. In another embodiment, the immunoprecipitation antibody is a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and an epitope between amino acids 219 and 226 of tau protein.
  • the immunoprecipitation antibody is a monoclonal antibody comprising immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively.
  • the immunoprecipitation antibody is a monoclonal antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 8, and a light chain variable region amino acid sequence of SEQ ID NO: 9. More particular, the immunoprecipitation antibody is PT9.
  • the isolated/ concentrated tau protein is digested with at least one enzyme before analyzing the digested peptides by liquid chromatography mass spectrometry (LC-MS) to detecting O-GlcN Acylation in the tau protein.
  • LC-MS liquid chromatography mass spectrometry
  • the isolated/ concentrated tau protein may be digested with trypsin, asp-N, and/or glu-C, to produce digested peptides.
  • the isolated tau protein may be divided into five (5) aliquots, which each aliquot digested under the following conditions: (1) digest with trypsin; (2) digest with asp-N; (3) digest with glu- C; (4) digest with trypsin followed by asp-N; and (5) digest with trypsin followed by glu-C.
  • the digested peptides are then subsequently analyzed by LC-MS, such as, for example, ultra-high performance liquid chromatography- MS/MS (UHPLC-MSMS), liquid chromatography - tandem mass spectrometry (LC -MS/MS), nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS) or nanoflow liquid chromatography - high resolution mass spectrometry (nLC- HRMS), to determine amounts of O-GlcN Acylated tau peptides in the biologic sample.
  • the LC- MS may analyze the digested peptides to determine a total amount of O-GlcN Acylated tau peptides in the sample.
  • the LC-MS may also analyze the digested peptide to determine an amount of S184/S185 O-GlcN Acylated tau peptides, an amount of SI 91 O-GlcN Acylated tau peptides, an amount of S208 ( -GlcN Acylated tau peptides, an amount of S400 O-GlcN Acylated tau peptides, or any combination thereof.
  • S184/S185 O-GlcN Acylated tau peptides may be detected by LC-MS by detecting presence of a polypeptide having a sequence of SEQ ID NO: 16 or 17.
  • SI 91 O-GlcN Acylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 18.
  • S208 O-GlcNAcylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 19.
  • S400 O-GlcN Acylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 20.
  • S 184/S 185 O-GlcN Acylated tau peptides may be detected by LC-MS by detecting ion transition from 600.3 m/z to 1001.5 m/z, 600.3 m/z to 996.5 m/z, or from 600.3 m/z to 798.4 m/z.
  • S191 (9-GlcN Acylated tau peptides may be detected by detecting ion transition from 538.9 m/z to 959.5 m/z, or 538.9 m/z to 756.4 m/z.
  • S208 (9-GlcN Acylated tau peptides may be detected by detecting ion transition from 798.9 m/z to 619.3 m/z, 798.9 m/z to 874.4 m/z, or from 798.9 m/z to 1115.5 m/z.
  • S400 O-GlcN Acylated tau peptides may be detected by detecting ion transition from 652.8 m/z to 1101.4 m/z.
  • an assay or method 100 for detecting O-GlcNAc-tau peptides from a biologic sample from a human subject is provided, as shown in Fig. 1.
  • the method 100 comprises a first step 102 for isolating and/or concentration the tau protein from the biologic sample by immunoprecipitation before analysis of the sample to detect any O-GlcNAcylation in the same manner as described above.
  • step 104 the O-GlcNAc sites of the isolated/concentrated tau protein are labeled with biotin to produce biotinylated O-GlcNAc peptides.
  • a N- azidoacetylgalactosamine (GalNAz) group is attached to the O-GlcNAc sites of the isolated/concentrated tau protein by reacting the isolated/concentrated tau with UDP-N- azidoacetylgalactosamine (UDP-GalNAz) and Gal-Tl (Y289L).
  • UDP-N- azidoacetylgalactosamine UDP-N- azidoacetylgalactosamine
  • Gal-Tl Y289L
  • the GalNAzylated and biotinylated O-GlcNAc tau protein is digested with trypsin, asp-N, and/or glu-C, to produce digested peptides.
  • the isolated tau protein may be divided into five (5) aliquots, which each aliquot digested under the following conditions: (1) digest with trypsin; (2) digest with asp-N; (3) digest with glu-C; (4) digest with trypsin followed by asp-N; and (5) digest with trypsin followed by glu-C.
  • the digested biotinylated peptides are immobilized to a solid support.
  • the digested biotinylated peptides are bound to streptavidin beads, preferably magnetic streptavidin beads, to immobilize the digested biotinylated peptides to the beads.
  • supernatant from the sample may be removed and thereby isolating and/or concentrating the digested biotinylated peptides.
  • step 110 the immobilized and digested biotinylated peptides are reacted with Na2SO3 in a P-elimination - Michael addition reaction to release sulfited peptides from the solid support.
  • the P-elimination - Michael addition reaction substitutes the GalNAzylated and biotinylated O-GlcNAc site with a sulfite group.
  • the sulfited peptides are analyzed using LC-MS to detect sulfite modification in at least one of residue 184, 185, 191, 208, and 400 of the tau protein.
  • the amount of sulfite modifications detected correspond to an amount of an amount of (9-GlcN Acylated tau peptides in the biologic sample.
  • the LC-MS may analyze the digested peptides to determine a total amount of sulfite modified tau peptides in the sample.
  • the LC-MS may also analyze the digested peptide to determine an amount of S184/S185 sulfite modified tau peptides, an amount of SI 91 sulfite modified tau peptides, an amount of S208 sulfite modified tau peptides, an amount of S400 sulfite modified tau peptides, or any combination thereof.
  • S184/S185 sulfite modified tau peptides may be detected by LC-MS by detecting presence of a polypeptide having a sequence of SEQ ID NO: 21 or 22.
  • SI 91 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 23.
  • S208 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 24.
  • S400 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 25.
  • the assays and methods of the present application measure O- GlcN Acylated tau peptides from a CSF sample from a human subject.
  • OGA inhibition is determined to be present in the brain of the subject when the amount of O-GlcN Acylated tau peptides is above a predetermined threshold value.
  • the assays and methods of the present application measure S184/S185 O-GlcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof from a CSF sample from a human subject and the presence of OGA inhibition is determined when an amount of S 184/S 185 O-GlcN Acylated tau peptides, S 191 O-GlcNAcylated tau peptides, S208 (9-GlcN Acylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof is above a predetermined threshold value.
  • the predetermined threshold values may be any suitable threshold value, e. g. , a suitable threshold value for distinguishing those subjects who have a decreased level of OGA inhibition as compared to those subjects who have increased level of OGA inhibition or who are healthy.
  • the predetermined threshold values may be determined as a total O-GlcN Acylated tau peptide concentration or a concentration of S184/S185 O-GlcN Acylated tau peptides, S191 O- GlcNAcylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof for differentiating those patients that have decreased levels of OGA inhibition and those who have increased levels.
  • Subjects identified as having a decreased level of OGA inhibition may be directed to obtain further clinical tests, such as, for example, PET imaging or other diagnostic tests, to further assess brain pathologies of these subjects.
  • subjects identified as having a decreased level of OGA inhibition may be administered an O-GlcNAc hydrolase inhibitor.
  • O-GlcNAc hydrolase inhibitors are described in Bartolome-Nebreda, et al., “O-GlcNAcase inhibitors as potential therapeutics for the treatment of Alzheimer’s disease and related taupathies: analysis of the patent literature,” Expert Opinion on Therapeutic Patents, Vol. 31, No. 12, 1117-1154 (2021), which is incorporated in its entirety by reference herein.
  • the O-GlcNAc hydrolase inhibitor is Thiamet G having the structure of Formula I: (Formula I) or a derivative thereof.
  • the O-GlcNAc hydrolase inhibitor is (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2- d]thiazole-6,7-diol (MK-8719), or N-[4-fluoro-5-[[2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]piperidin-l-yl]methyl]-l,3-thiazol-2-yl]acetamide (LY-3372689).
  • the assays and methods of the present application measure O- GlcNAcylated tau peptides from a CSF sample from a human subject and subsequently determines that the subject is need of ( -GlcN Ac hydrolase inhibition.
  • the assays and methods of the present application measure ( -GlcN Acylated tau peptides from a CSF sample from a human subject and subsequently determines that the subject is in need of O-GlcNAc hydrolase inhibition when an amount of S 184/S 185 G-GIcN Acylated tau peptides, S 191 G-GIcN Acylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 O-GlcNAcylated tau peptide, or any combination thereof is below a predetermined threshold value.
  • the predetermined threshold values may be any suitable threshold value, e.g., the predetermined threshold values may be determined as a total O- GlcNAcylated tau peptide concentration or a concentration of SI 84/S 185 G-GIcN Acylated tau peptides, SI 91 G-GIcN Acylated tau peptides, S208 G-GIcN Acylated tau peptides, S400 O- GlcNAcylated tau peptide, or any combination thereof for differentiating those patients that are healthy and those patients that have an G-GIcN Acylation modulated disease.
  • the predetermined threshold value may correspond to a baseline value or a value that is significantly higher than the baseline value.
  • “significantly higher” refers to a higher value that is statistically significant, not due to chance alone, which has a p-value of 0.05 or less.
  • “Significantly higher” can be at least about 1%, 2%, 5%, or 10% higher than that found in healthy subjects, at a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc.
  • the baseline value may correspond to a mean level in a population of healthy individuals.
  • the baseline value may also correspond to a mean value of previous levels determined in the same subject.
  • a method of the present application comprises (i) detecting O- GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcN Acylated tau peptides in a biologic sample obtained from the subject; and (ii) determining the effectiveness of an O-GlcNAc hydrolase inhibitor administered to the subject based a total amount of G-GIcN Acylated tau peptide or an amount of S 184/S 185 G-GIcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 O- GlcNAcylated tau peptide, or any combination thereof measured.
  • effectiveness of a treatment is determined by monitoring a total amount of O- GlcNAcylated tau peptide or an amount of SI 84/S 185 G-GIcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 O-GlcNAcylated tau peptide, or any combination thereof, before, during, and/or after administration of the treatment.
  • An increase in values relative to baseline signals an increase in G-GIcN Ac hydrolase inhibition.
  • the treatment can be treatment for a neurodegenerative disease, such as a tauopathy and/or amyloidogenic disease.
  • the tauopathy and/or amyloidogenic disease may be selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, sub
  • the tauopathy and/or amyloidogenic disease is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease), FTDP-17 or progressive supranuclear palsy.
  • the tauopathy and/or amyloidogenic disease is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease).
  • a method of the present application comprises (i) detecting O- GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcN Acylated tau peptides in a biologic sample obtained from the subject; and (ii) determining the effectiveness of treatment with an O-GlcNAc hydrolase inhibitor administered to the subject based on a total amount of O-GlcN Acylated tau peptide or an amount of S 184/S 185 (9-GlcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof measured.
  • effectiveness of treatment with an O-GlcNAc hydrolase inhibitor administered to the subject is determined by monitoring a total amount of O- GlcNAcylated tau peptide or an amount of SI 84/S 185 O-GlcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof, before, during, and/or after administration of the treatment.
  • An increase in values relative to baseline signals an increase in an O-GlcNAc hydrolase inhibition.
  • a method of the present application comprises (i) detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcNAcylated tau peptides in a biologic sample obtained from the subject; and (ii) determining whether or not the subject is suitable for treatment with an O-GlcNAc hydrolase inhibitor based on a total amount of O-GlcN Acylated tau peptide or an amount of S184/S185 O-GlcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O- GlcNAcylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof measured.
  • a subject is suitable for treatment with an O-GlcNAc hydrolase inhibitor if the total amount of O-GlcNAcylated tau peptide or the amount of S184/S 185 G-GIcN Acylated tau peptides, SI 91 (9-GlcN Acylated tau peptides, S208 O- GlcNAcylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof measured is lower than a pre-determined threshold value.
  • the method further comprises a step of administering an OGA inhibitor to a subject determined to be in need of OGA inhibition or to a subject determined to be suitable for treatment with an OGA inhibitor.
  • Example 1 provides an exemplary method for determining whether tau O- GlcNAcylated at S400 could be captured and detected with IP-LC-MSMS methodology using a total tau antibody.
  • a showing that IP-LC-MSMS methodology is capable of detecting tau O- GlcNAcylated at S400 demonstrates that the IP-LC-MSMS methodology can also be applied to identifying other tau (9-GlcN Acylation sites in mouse brain homogenates (BH).
  • Ammonium bicarbonate, formic acid (LA) 98-100 %, methanol (for spectroscopy), chloroform, Tween20, ammonia solution 25 % (Suprapur), sodium hydroxide (NaOH), 1 M HEPES and bovine serum albumin (BSA) Cohn fraction V were supplied by Merck. MilliQ water with a resistivity of 18.2 MQ.cm at 25 °C was generated with a MilliporeTM-purification system. Trifluoroacetic acid (TFA) and sodium sulfite (Na2SCh) were purchased from VWR. Acetonitrile (ULC-MS) was bought from Actu-All Chemicals.
  • H-SPVV-(O-GlcNAc)S-GDTSPR-OH (SEQ ID NO: 26) was supplied by Tebu Bio, H-SPWSGDTSPR-OH (SEQ ID NO: 27) by GenScript, and peptides H-TPSLPTPPTR-OH (SEQ ID NO: 28), H-TPSLP(pT)PPTR-OH (SEQ ID NO: 29), H- SR(pT)PSLP(pT)PPTREPK-OH (SEQ ID NO: 30), H-TPSLPTPPTR*-OH (SEQ ID NO: 31) and H-TPSLP(pT)PPTR*-OH ((SEQ ID NO: 32) (*: Arginine labelled with 13 C and 15 N) were purchased from New England Peptide.
  • Octyl P-D-glucopyranoside and Dulbecco's phosphate buffered saline (DPBS) were supplied by Thermo Scientific. Antibody PT9 was produced. Trypsin Gold, asp-N sequence grade and glu-C sequence grade were obtained from Promega.
  • Brains were rapidly harvested, immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. Brains were homogenized in buffer H in a 1:6 ratio (weight (mg):buffer volume (pL)), using Lysing matrix D tubes (MP Biomedicals #6913-500) in a Fastprep-24 Homogenizer (MP Biomedicals, 116005500 6.0 m/s, time 20 s). Homogenates were then centrifuged at 20,000 g, at room temperature, for 40 minutes to remove cell debris.
  • Non-labelled and stable isotopically labelled (SIL) peptide standard solutions individual stock solutions of 200 pM were prepared in water:acetonitrile:acetic acid (89: 10:1). The stock solutions were aliquoted and stored at -80°C. Standard working dilutions were freshly prepared for each batch of samples in 90:10:0.1 water: acetonitrile: formic acid.
  • SIL isotopically labelled
  • a method for concentrating tau and O-GlcNAc-tau protein from a biologic sample using immunoprecipitation (IP) was used to obtain tau extract from mice brain homogenates as described further below.
  • IP reaction 93.5 pL of Dynabeads protein G (Thermo Fisher Scientific) corresponding to 2.8 mg beads was washed two times in LoBind Eppendorf tubes (Eppendorf) with 125 pL 0.01% Tween20 in PBS. The tubes were placed in a DynaMagTM-2 Magnet (Thermo Fisher Scientific) and the supernatant was discarded.
  • IP immunoprecipitation
  • Buffer H high Salt buffer
  • 10 mM Tris Thermo Fisher #15567-027), 800 mM NaCl (Thermo Fisher #24740-011), 1 mMEDTA (Thermo Fisher #15575- 020), 10 % sucrose (Sigma #S9378), pH 7.4, filtered on 0.22 pm and supplemented with protease cocktail (Roche Complete mini EDTA free #11836170001) and phosphatase cocktail inhibitors (Roche Phostop #04906845001) as recommended by the manufacturer.
  • the samples were mixed and incubated overnight at 4°C while rotating on a hula mixer (Thermo Fisher Scientific). The supernatant was collected as immunodepleted fraction.
  • 150 pL 50 mM ammonium bicarbonate (pH 8) + 10% of 0.1% Tween20 in PBS
  • 50 mM ammonium bicarbonate (pH 8) + 10% of 0.1% Tween20 in PBS was added to the beads and subsequently vortex mixed and spun (Minispin Plus centrifuge, Eppendorf). The supernatant was collected as wash and the beads were resuspended in 150 pL of 50 mM ammonium bicarbonate, pH 8.
  • the samples were stored on melting ice while preparing reagents for trypsinization: trypsinization was started on the day IP was finished.
  • Trypsinization 15 JJ.L of acetonitrile was added to the beads + 150 pL of 50 mM ammonium bicarbonate, vortex mixed for 5 s and spun down for 15 s with a Minispin Plus centrifuge. 25 pL of 0.05 mg trypsin mL' 1 in 50 mM acetic acid was added and the samples were subsequently incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific) for on-bead digestion. Afterwards, the digestion was quenched by adding 15 pL of formic acid and briefly vortex mixing.
  • a solution containing approximately 3 ng/mL recombinant tau 2N4R (recombinant tau- 441 from Promise Proteomics) in 50 mM ammonium bicarbonate was digested in parallel with the biologic samples as positive control for trypsinization.
  • 150 pL 50 mM ammonium bicarbonate was digested under the same conditions as digestion method blank.
  • Ultra-high performance liquid chromatography- MS/MS analyses were performed on an ultra-high performance liquid chromatograph from Shimadzu consisting of 2 Nexera LC30AD liquid pumps set up to provide binary solvent gradients, a SIL-AC30 autosampler, a CTO-20AC column oven, a communications bus module (CBM-20A) and a sample Rack Changer II, hyphenated via a Turbo-IonsprayTM Interface (Sciex) to a 6500 triple quadrupole mass spectrometer (Sciex).
  • Analyst versions 1.6.3 and 1.7 (Sciex) was used as instrument control and data processing software.
  • 50 pL of digest was injected on a YMC-Triart Cl 8 column, 2.0 mm x 50 mm, 1.9 pm (YMC) and thermostatically (60 °C) eluted.
  • the mobile phase (MP) solvents consisted of 100:1 water + 0.05 % ammonia: acetonitrile (v:v) (A) and acetonitrile + 0.05 % ammonia (v:v) (B), and the gradient was set as follows (min/A%): 0.0/100, 5.0/70, 5.1/2, 6.6/2, 6.7/100, 10/100.
  • the flow rate was set at 0.5 mL/min.
  • the peptides were ionized with electrospray ionisation (ESI) in positive ion mode.
  • the ionspray voltage was set to 5500 V, temperature to 450°C, declustering potential to 86 V and entrance potential to 10 V.
  • Ion source gas 1, gas 2 and curtain gas were set to 50, 40 and 30, respectively.
  • CAD gas was set to 6.
  • Table 1 The selected MS transitions used for multiple reaction monitoring of the target peptides are provided in Table 1, which provides MRM-transitions used in UHPLC-MSMS experiments for the detection of tau S400 O-GlcNAcylation in mouse brain homogenate samples. Transitions were divided into two periods: period 1 from 0 to 2.6 min. and period 2 from 2.6 to 10 min. Data processing was performed with Analyst 1.7 or Sciex OS 1.6 (Sciex). Table 1.
  • a second set of surrogate peptides namely TPSLP(T217)PPTR (SEQ ID NO: 36), TPSLP(pT217)PPTR (SEQ ID NO: 34), and SR(pT)PSLP(pT217)PPTREPK (SEQ ID NO: 35), representing tau either non-modified or phosphorylated at or around T217, was used to monitor the influence of Thiamet G treatment on tau T217 phosphorylation. Tryptic peptide concentrations in the sample extracts were calculated using standard dilutions in solvent (no matrix matching).
  • Table 2 shows relative concentrations of monitored post-translational modifications of tau (S400 O-GlcNAcylation and phosphorylation in the mid-region (at and around T217)) in brain homogenates of P301S transgenic mice from 2 independent Thiamet G treatment studies (Study 1 and Study2), calculated as ‘concentration surrogate peptide with PTM * 100 / (concentration surrogate peptide with PTM + concentration non-modified surrogate peptide)’, expressed in %.
  • the data of Table 2 are obtained with IP-LC-MSMS (semi-quantitative data), with the results being averages of duplicates.
  • Example 2 describes an exemplary method for identifying O-GlcNAc-sites from O- GlcNAc rec htau produced in the manner described below. Identification of ( -GlcN Acylated tau peptides at relatively high concentrations present in an O-GlcNAc rec htau digest would allow gathering of high-quality LC-MSMS data, to be used as reference for identification of low concentration (9-GlcN Ac-peptides in mouse BH extracts in Example 4.
  • Example 2 Unless indicated otherwise below, the materials used in Example 2 are the same as described above or Example 1.
  • E. coli strain T7-Express (NEB) was transformed with the human MAPT-tau 2N4R and OGT encoding plasmids and double transformants selected on LB-Agar plates with 50 pg/mL carbenicillin and 25 pg/mL kanamycin.
  • colonies were used to set up expression cultures in 2.5 L Ultra Yield flasks (Thomson) containing 1 L LB medium, 50 .g carbenicillin and 25 pg/mL kanamycin, at 37°C and 300 RPM shaking. Once OD600nm reached 0.5, cultures were cooled to 22°C and expression induced with 0.5 mM IPTG overnight. In some cases, the LB medium was supplemented with 1 % glucose during expression. Next morning cells were harvested by centrifugation at 6000 RPM.
  • the beads were captured and washed by three rounds of centrifugation and re-suspension with 10 volumes buffer A supplemented with 30 mM Imidazole.
  • the beads were packed on an empty C-10 column connected to a AKTA Pure FPLC and washed to stable A280nm baseline with the same buffer.
  • Tau protein was eluted with buffer A supplemented with 500 mM Imidazole and peak fractions analysed by SDS-PAGE.
  • the purest fractions were pooled, concentrated, and loaded immediately on a Superdex 200 pg 16/600 SEC column, pre-equilibrated with SEC buffer (25 mM Tris, 150 mM NaCl, 1 mM DTT).
  • the column was run at 1.5 mL/min with SEC buffer, UV monitored at 280 nm and fractions collected. Peak fractions were analyzed by SDS-PAGE, purest fractions pooled, flash frozen and stored at -80°C as aliquots.
  • O- GlcNAcylated recombinant human tau (O-GlcNAc rec htau), produced in the manner described above, was digested with different enzymes, rendering 5 digestion conditions: (1) trypsin only, (2) asp-N only, (3) glu-C only, (4) trypsin followed by asp-N, and (5) trypsin followed by glu-C.
  • the O-GlcNAc rec htau samples were incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific) for digestion.
  • the digestion was quenched afterwards by adding 30 pL of formic acid.
  • digestion condition 4 19 pL of 0.004 mg asp-N mL’ 1 in 50 mM ammonium bicarbonate, was added and the mixtures were again incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific).
  • the valve was switched to elute the trapped analytes in backflush mode onto an EASY-Spray PepMap Cl 8 nLC column, 3 pm, 75 pm x 15 cm (Thermo Fisher Scientific).
  • the analytes were chromatographed with a linear gradient provided at 300 nL/min by a nLC binary pump, with mobile phases A: 100% water + 0.1% FA, and B: 20:80 water: acetonitrile + 0.1% FA. Details of the chromatographic conditions are provided in Table 4.
  • Table 4 provides LC-gradient and valve switching timing of the nLC-HRMS method used for analysis of (9-GlcNAcylated recombinant human tau digests.
  • Mobile phase solvents A and B are pumped by the nLC binary pump, mobile phase solvent C is provided by the pLC loading pump.
  • Valve position ‘O’ pLC flow passes via sample loop through the trap column and finally elutes to waste (load position).
  • Valve position ‘ 1’ nLC flow passes through trap column and analytical column to finally enter the MS system.
  • the column ‘Parameter’ indicates valve position (0 or 1), and percentage (%) B and C solvent, respectively.
  • centroid data were recorded with electrospray ionisation mode in positive polarity.
  • Full MS data were acquired with scan range set from m/z 375 to m/z 1,500.
  • Resolution in full MS mode was set to 60,000 FWHM, with Automatic Gain Control (AGC) target at 3e6 and maximum injection time of 60 ms.
  • AGC Automatic Gain Control
  • Polysiloxane was used for lock mass correction.
  • Resolution for ddMS2 was set to 15,000 with AGC target at le5 and maximum injection time of 50 ms (loop count 3 and topN set to 3).
  • the isolation window of the quadrupole was set to 1.6 m/z and scan range of ddMS2 experiments was set from m/z 200 to m/z 2,000. Normalized collision energy was set to 28. Data dependent fragmentation settings: minimum AGC target: 2e3, intensity treshold: 4e4, charge-state exclusion: unassigned, 1, 7, 8, >8, exclude isotopes: on, dynamic exclusion: 5 s.
  • XCalibur 3.0 (Thermo Fisher Scientific) was used for nLC-HRMS data acquisition and manual data processing.
  • Proteome Discoverer 2.3 (Thermo Fisher Scientific) was used to create a peak list from the raw data with ‘Fixed Value PSM Validator’ node, with the value for 'Peptide Confidence At Least' set to 'Medium'. Maximum missed cleavages was set to 2, minimum peptide length to 6 AAs, precursor mass mass accuracy: 10 ppm, fragment mass mass accuracy: 0.02 Da, and selected dynamic modification: HexNAc.
  • a FASTA-file with the protein sequence of human tau 2N4R isoform F of SEQ ID NO: 1 (Pl 0636 downloaded from uniprot.org) was used as protein database.
  • O-GlcNAc-peptides eluted just before -or co-eluted with- their non-modified counterpart, thereby giving additional confirmation of their identity.
  • Extracted ion chromatograms and MS fragmentation spectra of the below discussed peptide identifications are provided in Figs. 2-13.
  • the mass of the precursor ion non-modified peptide is at m/z 551.28038 (doubly charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 652.82006 (doubly charged).
  • HLSNVSSTGSI*O-GlcNAc peptides (peptide of SEQ ID NO: 38 having at least one O-GlcNAc site) originating from the C-terminus of tau were identified in a trypsin - asp-N digest of O-GlcNAc rec htau (with the same exact mass as SPW-(O-GlcNAc)S- GDTSPR(SEQ ID NO: 20)).
  • Fig. 3 shows MS fragmentation spectra of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20).
  • Diagnostic O-GlcNAc ions are predominantly lost upon fragmentation. Dotted arrows to the right side indicate product ions with O-GlcNAc modification.
  • Table 5 below identifies product ions of the SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) peptide, with the product ion annotation based on O-GlcNAcylation at S400. The product ions y7 and 78 are listed in bold to reflect those being product ions that contain O-GlcNAc.
  • FIG. 4 shows extracted ion chromatograms of peptides HLSNVSSTGSI (SEQ ID NO: 34) and HLSNVSSTGSI*O-GlcNAc isomers (5 ppm mass extraction window).
  • the mass of the precursor ion non-modified peptide is at m/z 551.28038
  • FIG. 5 shows MS fragmentation spectra of peptides HLSNVSSTGSI (SEQ ID NO: 34) and HLSNVSSTGSI*O-GlcNAc isomers. Diagnostic O-GlcNAc ions (indicated by solid arrows to the left side) are predominantly lost upon fragmentation.
  • Table 6 below identifies product ions of HLSNVSSTGSI*O-GlcNAc peptide eluting at 15.53 min.
  • Product ion annotation is based on HLSNVSSTGSI (SEQ ID NO: 34) due to in-source loss of O-GlcNAc-moiety.
  • the peptide SGYSSPGSPGTPG-(O-GlcNAc)S-R was identified based on several diagnostic product ions, locating the O-GlcNAcylation at S208 (based on 2N4R tau isoform): particularly the product ion at m/z 619.3047, originating from y4-fragmentation with retention of the O-GlcNAc-moiety, provided unambiguous confirmation.
  • FIG. 6 shows extracted ion chromatograms of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 14) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) (5 ppm mass extraction window).
  • the mass of the precursor ion non-modified peptide is at m/z 697.32076 (doubly charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 798.86045 (doubly charged).
  • the product ions y4, y7, y8, y9, ylO, yl l and y 12 are listed in bold to reflect those being product ions that contain O-GlcNAc.
  • the product ion originating from y4 fragmentation indicates O-GlcN Acylation at S208.
  • TPPSSGEPPK-(O-GlcNAc)S-GDR SEQ ID NO: 18
  • TPPSSGEPPK-(O-GlcNAc)S-GDR SEQ ID NO: 18
  • Fig. 8 shows extracted ion chromatograms of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18)(5 ppm mass extraction window).
  • Fig. 9 shows MS fragmentation spectra of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO:
  • TPPSSGEPPK (SEQ ID NO: 12), but not containing S 191 , was detected with O-GlcNAcylation.
  • TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18)
  • broad chromatographic peak shapes were obtained for both the unmodified and O-GlcNAc peptide, matching well however in terms of shape and retention.
  • Fig. 10 provides extracted ion chromatograms of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc representing tau O-GlcNAcylation at either S 184 or S 185 (SEQ ID NO: 16 or 17) (5 ppm mass extraction window).
  • Fig. 11 shows MS fragmentation spectra of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) (obtained with PRM). Diagnostic O-GlcNAc ions (indicated by solid arrows to the left) are predominantly lost upon fragmentation. Dotted arrows to the right indicate product ions with O-GlcNAc modification.
  • Table 9 identifies product ions of TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) peptide.
  • the product ion y8 is listed in bold to reflect it containing O-GlcNAc.
  • Product ion annotation based is based on O-GlcNAcylation at SI 85.
  • a long tryptic peptide containing T123 and corresponding to sequence QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHV-T123-QAR (SEQ ID NO: 39) was detected with O-GlcNAcylation: the O-GlcN Acylated peptide eluted just before its unmodified form and signature peptides for O-GlcN Acylation (m/z 204, etc.) were observed.
  • Product ions in accordance with the peptide sequence were detected, however all without an (9-GlcN Ac-moiety.
  • Fig. 12 shows extracted ion chromatograms of peptides QAAAQPHTEIPEGTTAE EAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAG HVTQAR*O-GlcNAc.
  • the mass of the precursor ion non-modified peptide is at m/z 989.71897 (quadruply charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 1040.48881 (quadruply charged).
  • Fig. 12 shows extracted ion chromatograms of peptides QAAAQPHTEIPEGTTAE EAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAG HVTQAR*O-GlcNAc.
  • the mass of the precursor ion non-modified peptide is
  • Example 4 Based on the data generated in Example 2, the three following peptides were selected to for further analysis in mouse BH in Example 4 discussed below: SGYSSPGSPGTPG-(O- GlcNAc)S208-R (SEQ ID NO: 19), TPPSSGEPPK-(O-GlcNAc)S191-GDR(SEQ ID NO: 18) and TPPSSGEPPK*O-GlcNAc (at S184 or S185, most probably O-GlcNAcylated at S185)(SEQ ID NO: 16 or 17).
  • Example 3 describes an exemplary method for selective clean-up and derivatization of O-GlcNAc rec htau in an effort to mitigate matrix effects from highly abundant unmodified peptides in tryptic digests, and to improve position identification by mitigating the easy loss of O- GlcNAc-moieties by in-source fragmentation.
  • 0-elimination/Michael addition is used in Example 3 for the structural analysis of the O-glycosylated tau proteins.
  • a process for 0- elimination/Michael addition of recombinant human tau protein is described below.
  • Example 3 Unless indicated otherwise below, the materials used in Example 3 are the same as described above or Example 1.
  • O-GlcNAc sites of O- GlcNAc rec htau were first chemo- enzymatically labelled with biotin, biotinylated O-GlcNAc rec htau was subsequently trypsinized, followed by immobilization/purification of biotinylated O- GlcNAc-peptides with streptavidin beads. Non-bound peptides were removed by washing the beads. Immobilized O-GlcNAc-peptides were released by transformation into free sulfited peptides via P-elimination - Michael addition reaction.
  • a schematic representation of the derivatization and purification process 200 described below is provided in Figure 2.
  • the upper aqueous phase was then carefully removed as much as possible and discarded while leaving the interface layer containing the protein precipitate intact.
  • 450 pL of methanol was added, the tube was vortex mixed and then centrifuged for 5 minutes at 18,000/g to pellet the protein. The supernatant was removed and discarded, and the procedure was repeated once more. The pellet was allowed to air-dry.
  • the protein was resuspended in 40 pL of 1% SDS in 20 mM HEPES pH 7.9, heated for 8 min at 90°C while shaking (ThermoMixer, Thermo Fisher Scientific) to dissolve the pellet, and immediately cooled on melting ice.
  • step 204 enzymatic labeling of O-GlcNAc rec htau with UDP-N- azidoacetylgalactosamine (UDP-GalNAz) and Gal-Tl (Y289L) was carried out by using the ‘Click-iTTM O-GlcNAc Enzymatic Labeling System’-kit (MP 33368, Thermo Fisher Scientific). After GalNAz-addition by overnight incubation at 4°C, modified O-GlcNAc rec htau was precipitated with the above chloroform/methanol/water clean-up. The protein pellet was dissolved in 50 pL 50 mM Tris pH 8.0 + 1% octyl P-D-glucopyranoside by heating for 8 min at 90°C while shaking, and immediately cooled on melting ice.
  • UDP-N- azidoacetylgalactosamine UDP-N- azidoacetylgalactosamine
  • Gal-Tl Y
  • step 206 iotinylation was performed via Click-iTTM azide/alkyne reaction by following the ‘Click- it Protein Analysis Detection Kits ’-protocol (MP 33372, Thermo Fisher Scientific). After incubating the reaction mixture for 30 min at room temperature, the GalNAzylated and biotinylated O-GlcNAc rec htau was precipitated with the above chloroform/methanol/water clean-up. The pellet was dissolved by heating for 8 min at 90°C while shaking in 150 pL 50 mM ammonium bicarbonate. After cooling on melting ice, 10 pL of acetonitrile was added followed by briefly vortex mixing.
  • step 208 For digestion in step 208, 25 pL of 0.05 mg trypsin/mL in 50 mM acetic acid was added and the sample was subsequently incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific).
  • the digest was incubated with DynabeadsTM M-280 Streptavidin (Thermo Fisher Scientific): 158 pL of 10 mg beads/mL in PBS pH 7.4 with 0.1% BSA and 0.02% sodium azide was aliquoted in a LoBind Eppendorf tube, placed on a DynaMagTM-2 Magnet, solvent was removed and the beads were washed in with 1 mL of DPBS + 0.1% octyl P-D-glucopyranoside by vortex mixing for 5 s. The tube was spun down and placed in a DynaMagTM-2 Magnet and the supernatant was discarded.
  • DynabeadsTM M-280 Streptavidin Thermo Fisher Scientific
  • the rec htau digest containing biotinylated GalNAz-O-GclNAc-peptides was evaporated with nitrogen gas at 45°C (not to completely dry), dissolved in 200 pL DPBS and transferred to the streptavidin beads. The mixture was incubated at room temperature for 30 min using gentle rotation. The tube was placed on a magnet and the depleted fraction was transferred and stored for analysis. The coated beads were washed four times with 400 pL DPBS + 0.1% BSA and once more with 400 pL DPBS.
  • step 212 P-eliminati on -Michael addition reaction was performed to release derivatized O-GlcNAc tryptic tau peptides as sulfited peptides: DPBS was discarded, 100 pL of 0.1 N NaOH and 0.6 M Na2SCh were added, followed by incubation of the beads at room temperature for 24h. The reaction was stopped by adding 50 pL of 0.3 M acetic acid.
  • the peptides were ionized with electrospray ionisation (ESI) in positive ion mode.
  • ESI electrospray ionisation
  • the ionspray voltage was set to 4500 V, temperature to 400°C, declustering potential to 80 V.
  • Ion source gas 1, gas 2 and curtain gas were set to 50, 40 and 30, respectively.
  • CAD gas was set to 6.
  • Data were acquired in TOF MS-mode with m/z-range set to measure from mlz 100 to mlz 1500. Table 11 below shows the exact mass and charge states of each of the intermediates of the purification process of the derivatization and purification process of Fig. 14.
  • SPWSGDTSPR SEQ ID NO: 15
  • SPW-(O-GlcNAc)S-GDTSPR SEQ ID NO: 20
  • SPW-(O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR (20 mDa mass extraction window) in the extract of GalNAzylated, biotinylated and trypsinized O- GlcNAc rec htau.
  • SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR was also observed in Fig.
  • FIG. 16 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S- GDTSPR (SEQ ID NO: 20), SPW-(O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc- GalNAz-biotin)S-GDTSPR (20 mDa mass extraction window) in the depleted fraction after capture of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau peptides with streptavidin beads.
  • Fig. 17 shows an extracted ion chromatogram of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25)(6 ppm mass extraction window).
  • the mass of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) is at m/z 583.26133 (doubly charged).
  • Fig. 18 shows MS fragmentation spectrum of SPW- (SO3)S-GDTSPR (SEQ ID NO: 25). Dotted arrows indicate product ions with sulfite modification.
  • Table 12 identifies product ions of the SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) peptide.
  • the product ions b5, b7, y7, y8 and y9 are listed in bold to reflect those being product ions that contain sulfite moiety.
  • Fig. 19 shows MS fragmentation spectrum of SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24).
  • the dotted arrows indicate product ions with sulfite modification.
  • the mass of SGYSSPGSPGTPG-(SO 3 )S-R (SEQ ID NO: 24) is at m/z 729.30171 (doubly charged).
  • Table 13 identifies product ions of the SGYSSPGSPGTPG-(SO 3 )S-R (SEQ ID NO: 24) peptide.
  • the product ions y2, y3, y4, y7-H2O, y7, ylO-H20 and ylO are listed in bold to reflect those being product ions that contain sulfite moiety.
  • Fig. 20 shows MS fragmentation spectrum of TPPSSGEPPK-(SO 3 )S-GDR (SEQ ID NO: 23).
  • the dotted arrows indicate product ions with sulfite modification.
  • the mass for TPPSSGEPPK-(SO 3 )S-GDR (SEQ ID NO: 23) is at m/z 738.32519 (doubly charged).
  • Table 14 identifies product ions of the TPPSSGEPPK-(SO 3 )S-GDR (SEQ ID NO: 23) peptide.
  • the product ions y4, yl2, yl3, y6, yl4, y7 and yl2 are listed in bold to reflect those being product ions that contain sulfite moiety.
  • Example 4 analyzed mouse BH digests without 0- elimination/Michael addition derivatization.
  • Example 4 IP-LC-MSMS of tau U-GIcN Acylation Site in Mouse Brain Homogenates [00171]
  • the mouse homogenates obtained in Example 1 are enzymatically digested according to Example 2 and further analyzed using UHPLC-MSMS and nLC-MSMS according to the methods below. Unless indicated otherwise below, the materials used in Example 4 are the same as described above or Example 1. The same mouse homogenates obtained in Example 1 are further analyzed in Example 4 according to the methods described below.
  • a 10-port switching valve was used to divert the eluate of the trap column to waste. After 6 min of sample loading, the valve was switched to elute the trapped analytes in backflush mode onto a nanoEase MZ HSS T3 Column, 100 A, 1.8 pm, 75 pm x 150 mm (Waters). The analytes were chromatographed with a linear gradient at 300 nL/min provided by a nLC binary pump, with mobile phases A: water + 0.1% FA, and B: 20:80:0.1 water:acetonitrile:FA. Details of the chromatographic conditions are provided in Table 16.
  • Table 16 provides LC-gradient and valve switching timing of the nLC-MSMS method used for analysis of O-GlcNAcylated tryptic tau peptides in extracts of mouse brain homogenates.
  • Mobile phase solvents A and B are pumped by the nLC binary pump, mobile phase solvent C is provided by the pLC loading pump.
  • Valve position ‘O’ pLC flow passes via sample loop through the trap column and finally elutes to waste (load position).
  • Valve position ‘1 ’ nLC flow passes through trap column and analytical column to finally enter the MS system.
  • the column ‘Parameter’ indicates valve position (0 or 1), and percentage (%) B and C solvent, respectively.
  • Table 17 lists MRM-transitions used in nLC-MSMS experiments for the detection of new O-GlcNAcylation sites in mouse brain homogenate samples. Table 17.
  • Fig. 21A shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either S 184 or S 185, originating from IP-ed and digested tau from a brain homogenate of Thiamet-G treated mice.
  • Fig. 21B shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85, originating from IP-ed and digested tau from a brain homogenate of non-treated mice.
  • Fig. 21A shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85, originating from IP-ed and digested tau from a brain homogenate of non-treated mice.
  • 21 C shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85, originating from a digest of O-GlcNAc recombinant human tau.
  • FIG. 22A shows nLC-MSMS chromatograms of SPW-((0-GlcNAc)S400)-GDTSPR and SPWSGDTSPR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G.
  • Fig. 22B shows nLC-MSMS chromatograms of SGYSSPGSPGTPG-(O-GlcNAc)S208-R and SGYSSPGSPGTPGSR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G.
  • FIG. 22C shows nLC-MSMS chromatograms of TPPSSGEPPK-(O-GlcNAc)S191-GDR and TPPSSGEPPKSGDR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G.
  • Fig. 22D shows nLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc (most probably O-GlcNAcylated at SI 85) and TPPSSGEPPK originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G.
  • Table 18 identifies peak areas of monitored surrogate tryptic peptides of tau, with and without O-GlcN Acylation, detected in brain homogenates of P301S transgenic mice from 2 independent Thiamet G treatment studies (Study 1 and Study 2), a method blank (IP-ed and digested buffer H) and in a brain homogenate of tau KO mice based on data obtained using IP-nLC-MSMS.
  • TPPSSGEPPK*O-GlcNAc most probably O-GlcN Acylated at S185 (see Table 18).
  • IP-nLC-MSMS analysis of BH extracts of transgenic P301S mice treated with Thiamet G enabled identification of low levels of O-GlcNAcylation in the N-terminal and mid domain of tau, namely at positions S208, S191 and S184 or S185.
  • SEQ ID NO: 32 Amino acids 212-221 of human tau protein phosphorylated at residue 217 (*: Arginine labelled with 13 C and
  • SEQ ID NO: 35 Amino acids 21-241 of human tau protein 2N4R phosphorylated at residues 212 and 217
  • SEQ ID NO: 36 Amino acids 212-221 of human tau protein 2N4R

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A method for detecting OGA inhibition in a brain of a subject. The method comprises detecting O-GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcNAcylated tau peptides in a biologic sample obtained from the subject. The method further comprises determining the presence of OGA inhibition in the brain of the subject when the amount of O-GlcNAcylated tau peptides is above a predetermined threshold value.

Description

MASS SPECTROSCOPY ASSAY FOR DETECTING O-P-LINKED N- ACETYLGLUCOSAMINYLATED TAU PEPTIDES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/374,132, filed August 31, 2022, the disclosure of which is herein incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The sequence listing of the present application is submitted electronically via The United States Patent and Trademark Center Patent Center as an XML formatted sequence listing with a file name “JAB7172WOPCTl_SEQLIST.xml”, creation date of August 29, 2023, and a size of 43 kilobytes (KB). This sequence listing submitted is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to methods for detecting neurodegeneration. In particular, the invention relates to methods of measuring an amount of O-0-linked N-acetylglucosaminylated tau (O-GlcNAc-tau) protein species in a biologic sample and uses thereof.
BACKGROUND OF INVENTION
[0004] Alzheimer’s Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in ethnic groups worldwide and presents a major present and future public health problem.
[0005] The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Neurofibrillary tangles are primarily composed of aggregates of hyper-phosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization. The binding of tau to microtubules takes place by ionic interactions between positive charges in the microtubule binding region of tau and negative charges on the microtubule lattice (Butner and Kirschner, J Cell Biol. 115(3):717-30, 1991). Tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the primary function of tau. Tau that is bound to the axonal microtubule lattice is in a hypo-phosphorylation state, while aggregated tau in AD is hyper-phosphorylated, providing unique epitopes that are distinct from the physiologically active pool of tau (Iqbal et al., Curr Alzheimer Res. 7(8): 656-664, 2010).
[0006] O-0-linked N-acetylglucosaminylation (O-GlcNAcylation), regulated by two antagonist enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), modulates tau phosphorylation and slows down its aggregation in vitro (Cantrelle et al., Frontiers in Molecular Neuroscience, 14: 661368, 2021). Therefore, the level of OGA modulation may be indirectly related to the process involving the (9-GlcN Ac-mediated regulation of enzymes implicated in phosphorylation dynamics or other actors in tau pathology (Cantrelle et al., 2021). However, there are a number of challenges in determining the level of OGA modulation. Identification of (9-GlcN Ac-sites is often hampered due to low stoichiometry. Generally, glycoproteins are enriched using lectins, antibodies, and/or solid-phase extraction (Kim, Molecules, 16:1987-2022, 2011; Calle et al., J. Am. Soc. Mass Spectrom. , 32: 2366-2375, 2021). Commonly used O-GlcNAc binding lectin such as wheat-germ agglutinin only bind weakly to monosaccharide O-GlcNAc: the use of these probes suffers from the contamination with nonspecific binding partners. In addition, since these probes bind preferentially with high abundance proteins or those with multiple clustered O-GlcNAc residues, the detection and isolation of low abundance proteins with single O-GlcNAc are often elusive (Kim, 2011). Alternatively, O- GlcNAc antibodies have been implemented as well, although the binding affinity of these antibodies to O-GlcNAc is relatively low, which prevents stringent washing conditions required for reducing non-specific interactions (Kim, 2011). Moreover, the concentration of tau in cerebrospinal fluid (CSF) is three orders of magnitude lower than in the brain (Sato et al., Neuron, 97:1284-1298, 2018). In addition, tau species found in CSF differ from tau in brain: the predominant forms of tau (99.9%) in CSF are C-terminally truncated containing the mid-domain but lacking the microtubule binding region and C-terminus, with cleavage between amino acid (AA) residues 222 and 225 (based on tau isoform 2N4R)(Sato, 2018).
BRIEF SUMMARY OF THE INVENTION
[0007] One exemplary embodiment of the present invention is directed to a method for detecting OGA inhibition in a brain of a subject. An OGA inhibitor may have been administered to the subject. The method comprises step a) detecting O-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample (e.g., cerebrospinal fluid (CSF)) obtained from the subject. OGA inhibition in the brain of the subject is determined to be present when the amount of O- GlcNAcylated tau peptides is above a predetermined threshold value. In some examples, the tau protein in the biologic sample is concentrated by immunoprecipitation before the detecting of O- GlcNAcylation.
[0008] In one example, step a) comprises detecting (9-GlcN Acylation in (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample obtained from the subject. In another example, step a) comprises detecting O-GlcNAcylation in at least one of residue 184, 185, 191, and 208 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample obtained from the subject. In a further example, step a) comprises detecting O- GlcNAcylation in (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 of the tau protein to determine an amount of O-GlcNAcylated tau peptides in a biologic sample obtained from the subject.
[0009] In one embodiment, the O-GlcNAcylation is detected by liquid chromatography mass spectrometry (LC-MS), such as nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS) or ultra-high performance liquid chromatography- MS/MS (UHPLC- MSMS). In another embodiment, the (9-GlcN Acylation is detected by an immunoassay. In some embodiments, the immunoassay is an ultrasensitive assay, such as a single-molecule array (SIMOA) assay.
[0010] An assay method of detecting O-GlcNAc-tau peptides is also provided. The method comprises obtaining a biologic sample from a human subject, and contacting the biologic sample (e.g., CSF) with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support (e.g., magnetic beads) to isolate tau protein from the biologic sample. The method further comprises digesting the isolated tau protein with at least one enzyme, and detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry (e.g., nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS)) to determine an amount of O-GlcNAcylated tau peptides in the biologic sample. In one example, (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 are detected. In another example, (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400 are detected.
[0011] The immunoprecipitation antibody used in the assay method binds to an epitope between amino acids 163 to 174 of human tau protein or an epitope between amino acids 219 to 226 of the human tau protein. For example, the immunoprecipitation antibody comprises an immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 comprising the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively and an immunoglobulin light chain LCDR1, LCDR2 and LCDR3 comprising the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively. Specifically, the immunoprecipitation antibody comprises a variable heavy chain region (VH) comprising the polypeptide sequence of SEQ ID NO: 8 and a variable light chain region (VL) comprising the polypeptide sequence of SEQ ID NO: 9.
[0012] In one embodiment, the assay method digests the isolated tau protein with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested peptides. For example, the isolated tau protein is divided into at least five aliquots and digested with the at least one enzyme, wherein the aliquots comprise: (1) an aliquot digested with trypsin; (2) an aliquot digested with asp-N; (3) an aliquot digested with glu-C; (4) an aliquot digested with trypsin followed by asp-N; and (5) an aliquot digested with trypsin followed by glu-C.
[0013] The LC-MS (e.g. , nLC-HRMS) may be used to detect O-GlcN Acylation at residue 184 or 185 by detecting presence of a polypeptide having a sequence of SEQ ID NO: 16 or 17. In particular, ( -GlcN Acylation may be detected at residue 184 or 185 is detected by detecting ion transition from 600.3 m/z to 1001.5 m/z, 600.3 m/z to 996.5 m/z, or from 600.3 m/z to 798.4 m/z. O-GlcN Acylation at residue 191 may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 18. In particular, (9-GlcN Acylation at residue 191 may be detected by detecting ion transition from 538.9 m/z to 959.5 m/z, or 538.9 m/z to 756.4 m/z. O-GlcNAcylation may be detected at residue 208 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 19. In particular, (9-GlcN Acylation at (ii) residue 208 is detected by detecting ion transition from 798.9 m/z to 619.3 m/z, 798.9 m/z to 874.4 m/z, or from 798.9 m/z to 1115.5 m/z. O-GlcN Acylation may be detected at residue 400 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 20. In particular, O-GlcNAcylation at residue 400 may be detected by detecting ion transition from 652.8 m/z to 1101.4 m/z.
[0014] In another aspect of the present application, another assay method of detecting O- GlcNAc-tau peptides is provided. This alternative method comprises obtaining a biologic sample (c.g, CSF) from a human subject and contacting the biologic sample with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support to isolate tau protein from the biologic sample. The method also comprises labelling O-GlcNAc sites of the isolated tau protein with biotin to produce biotinylated O-GlcNAc peptides, digesting the biotinylated O-GlcNAc peptides with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested biotinylated peptides, and immobilizing the digested biotinylated peptides to a solid support ( .g, streptavidin beads). The method further comprises reacting the digested biotinylated peptides with Na2SCh in a P-elimination - Michael addition reaction to release sulfited peptides from the solid support. The method further comprises detecting sulfite modification in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry to determine an amount of G-GlcN Acylated tau peptides in the biologic sample.
[0015] These and other aspects of the invention will become apparent to those skilled in the art after a reading of the following detailed description of the invention, including the figures and appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0016] Fig. 1 shows a method for detecting O-GlcNAc-tau peptides according to an exemplary embodiment of the present application.
[0017] Fig. 2 shows extracted ion chromatograms of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0018] Fig. 3 shows MS fragmentation spectra of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0019] Fig. 4 shows extracted ion chromatograms of peptides HLSNVSSTGSI (SEQ ID NO: 36) and HLSNVSSTGSI*O-GlcNAc isomers generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0020] Fig. 5 shows MS fragmentation spectra of peptides HLSNVSSTGSI (SEQ ID NO: 36) and HLSNVSSTGSI*O-GlcNAc isomers generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0021] Fig. 6 shows extracted ion chromatograms of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 15) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0022] Fig. 7 shows MS fragmentation spectra of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 15) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0023] Fig. 8 shows extracted ion chromatograms of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0024] Fig. 9 shows MS fragmentation spectra of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2. [0025] Fig. 10 shows extracted ion chromatograms of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0026] Fig. 11 shows MS fragmentation spectra of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0027] Fig. 12 shows extracted ion chromatograms of peptides QAAAQPHTEIPEGTTAE EAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGI GDTPSLEDEAAGHVTQAR*O-GlcNAc generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0028] Fig. 13 shows MS fragmentation spectra of peptides QAAAQPTHEIPEGTTAEEAGI GDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGDTPSL EDEAAGHVT QAR*O-GlcNAc generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 2.
[0029] Fig. 14 another method for detecting O-GlcNAc-tau peptides according to an exemplary embodiment of the present application described in Example 3.
[0030] Fig. 15 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), SPW- (O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR in the extract of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau generated according to an exemplary embodiment of the present application described in Example 3.
[0031] Fig. 16 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), SPW- (O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR in the depleted fraction after capture of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau peptides with streptavidin beads generated according to an exemplary embodiment of the present application described in Example 3. [0032] Fig. 17 an extracted ion chromatogram of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
[0033] Fig. 18 MS fragmentation spectrum of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
[0034] Fig. 19 shows MS fragmentation spectrum of SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
[0035] Fig. 20 shows MS fragmentation spectrum of TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 3.
[0036] Fig. 21A shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of Thiamet-G treated mice according to an exemplary embodiment of the present application described in Example 4.
[0037] Fig. 21B shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of Thiamet-G treated mice according to an exemplary embodiment of the present application described in Example 4.
[0038] Fig. 21C shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of recombinant human tau according to an exemplary embodiment of the present application described in Example 4. [0039] Fig. 22A shows nLC-MSMS chromatograms of SPVV-((O-GlcNAc)S)-GDTSPR (SEQ ID NO: 20) and SPVVSGDTSPR (SEQ ID NO: 15) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
[0040] Fig. 22B shows nLC-MSMS chromatograms of SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) and SGYSSPGSPGTPGSR (SEQ ID NO: 14) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
[0041] Fig. 22C shows nLC-MSMS chromatograms of TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) and TPPSSGEPPKSGDR (SEQ ID NO: 13) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
[0042] Fig. 22D shows nLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17) and TPPSSGEPPK (SEQ ID NO: 12) generated by immunoprecipitation, enzyme digestion, and mass spectrometry analysis of a brain homogenate of mice treated with a high dose of Thiamet-G according to an exemplary embodiment of the present application described in Example 4.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It is noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
[0044] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
[0045] As used herein, the term “antibody” or “immunoglobulin” refers to a specific protein capable of binding an antigen or portion thereof. These terms are used herein in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies (including murine, human, human-adapted, humanized and chimeric monoclonal antibodies) and antibody fragments.
[0046] In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the present application can be of any of the five major classes or corresponding sub-classes. Preferably, the antibodies of the present application are IgGl , IgG2, IgG3 or IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the present application can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the present application include heavy and/or light chain constant regions from mouse antibodies or human antibodies.
[0047] In addition to the heavy and light constant domains, antibodies contain light and heavy chain variable regions. An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by “antigen-binding sites.” The antigen-binding sites are defined using various terms and numbering schemes as follows:
(i) Kabat: “Complementarity Determining Regions” or “CDRs” are based on sequence variability (Wu and Kabat, J Exp Med. 132:211-50, 1970). Generally, the antigen-binding site has three CDRs in each variable region (e.g., HCDR1, HCDR2 and HCDR3 in the heavy chain variable region (VH) and LCDR1, LCDR2 and LCDR3 in the light chain variable region (VL)); (ii) Chothia: The term “hypervariable region,” “HVR” refers to the regions of an antibody variable domain which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, J Mol Biol. 196:901-17, 1987). Generally, the antigen-binding site has three hypervariable regions in each VH (Hl, H2, H3) and VL (LI, L2, L3). Numbering systems as well as annotation of CDRs and HVRs have been revised by Abhinandan and Martin (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008);
(iii) IMGT: Another definition of the regions that form the antigen-binding site has been proposed by Lefranc (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003) based on the comparison of V domains from immunoglobulins and T-cell receptors. The International ImMunoGeneTics (IMGT) database (http:_//www_imgt_org) provides a standardized numbering and definition of these regions. The correspondence between CDRs, HVRs and IMGT delineations is described in Lefranc et al., 2003, Id.,-
(iv) The antigen-binding site can also be delineated based on “Specificity Determining Residue Usage” (SDRU) (Almagro, Mol Recognit. 17: 132-43, 2004), where SDR, refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact.
[0048] ‘Framework” or “framework sequence” is the remaining sequences within the variable region of an antibody other than those defined to be antigen-binding site sequences. Because the exact definition of an antigen-binding site can be determined by various delineations as described above, the exact framework sequence depends on the definition of the antigen-binding site. The framework regions (FRs) are the more highly conserved portions of variable domains. The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively) which generally adopt a beta-sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817, 1992; Tramontane et al., J. Mol. Biol. 215:175-182, 1990). Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical structures.” These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main- chain conformations.
[0049] As used herein, the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a bispecific or multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the constant region of the heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab’).
[0050] As used herein, the term “epitope” refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof, specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
[0051] As used herein, the term “tau” or “tau protein” refers to an abundant central and peripheral nervous system protein having multiple isoforms. In the human central nervous system (CNS), six major tau isoforms ranging in size from 352 to 441 amino acids in length exist due to alternative splicing (Hanger et al., Trends Mol Med. 15: 112-9, 2009). The isoforms differ from each other by the regulated inclusion of 0-2 N-terminal inserts, and 3 or 4 tandemly arranged microtubule-binding repeats, and are referred to as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R. As used herein, the term “control tau” refers to the tau 2N4R isoform of SEQ ID NO: 1 that is devoid of phosphorylation and other post-translational modifications. As used herein, the term “tau” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full-length wild type tau. The term “tau” also encompasses post- translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, ( -GlcN Acylation or phosphorylation.
[0052] Unless otherwise indicated, as used herein, the numbering of the amino acid in a tau protein or fragment thereof is with reference to the amino acid sequence set forth in SEQ ID NO: 1.
[0053] As used herein, the term “O-GlcNAcylated tau peptides,” “O-GlcNAcylated tau,” or “O-GlcNAcylated tau protein” means human tau protein or tau fragment that is GlcNAcylated at one or more residue of tau protein.
[0054] As used herein, the term “S184/S185 O-GIcN Acylated tau peptide,” “S184/S185 O- GlcNAcylated tau,” or “S184/S185 O-GIcN Acylated tau protein” means a human tau protein or tau fragment that is GlcNAcylated at residue 184 (SI 84) or residue 185 (SI 85) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0055] As used herein, the term “S 191 O-GIcN Acylated tau peptide,” “S 191 O-GIcN Acylated tau,” or “SI 91 O-GlcN Acylated tau protein” means a human tau protein or tau fragment that is GlcNAcylated at residue 191 (SI 91) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0056] As used herein, the term “S208 O-GlcNAcylated tau peptide,” “S208 O-GlcNAcylated tau,” or “S208 O-GIcN Acylated tau protein” means a human tau protein or tau fragment that is GlcNAcylated at residue 208 (S208) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0057] As used herein, the term “S400 O-GlcNAcylated tau peptide,” “S400 O-GlcNAcylated tau,” or “S400 O-GIcN Acylated tau protein” means a human tau protein or tau fragment that is GlcNAcylated at residue 400 (S400) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0058] As used herein, the term “S 184/S185 sulfite modified tau peptide,” “S184/S185 O- sulfite modified tau,” or “S184/S185 sulfite modified tau protein” means a human tau protein or tau fragment that is sulfite modified at residue 184 (SI 84) or residue 185 (SI 85) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0059] As used herein, the term “SI 91 sulfite modified tau peptide,” “S191 sulfite modified tau,” or “SI 91 sulfite modified tau protein” means a human tau protein or tau fragment that is sulfite modified at residue 191 (SI 91) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0060] As used herein, the term “S208 sulfite modified tau peptide,” “S208 sulfite modified tau,” or “S208 sulfite modified tau protein” means a human tau protein or tau fragment that is sulfite modified at residue 208 (S208) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0061] As used herein, the term “S400 sulfite modified tau peptide,” “S400 sulfite modified tau,” or “S400 sulfite modified tau protein” means a human tau protein or tau fragment that is sulfite modified at residue 400 (S400) of tau protein, wherein the numbering of the positions is according to the numbering in SEQ ID NO: 1.
[0062] As used herein, the term “immunoprecipitation antibody” refers to an antibody that binds to an antigen of interest and is or becomes directly or indirectly linked to a solid support. Examples of solid supports include, but are not limited to, microparticles or beads, such as magnetic beads, paramagnetic beads, agarose beads, or streptavidin beads. Examples of an immunoprecipitation antibodies include, but are not limited to, a monoclonal antibody that binds to tau. In particular, the immunoprecipitation antibody does not bind to the N-terminal or the mid domain regions of the tau protein. More particularly, the immunoprecipitation antibody binds to tau in the proline rich region of the tau protein.
[0063] The immunoprecipitation antibody can be a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and/or amino acids 219 and 226 of tau protein, and the numbering of the positions is according to the numbering in SEQ ID NO: 1. According to embodiments of the present application, the immunoprecipitation antibody can be a monoclonal antibody comprising immunoglobulin heavy chain HCDR1 , HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7. In a particular embodiment, the immunoprecipitation antibody is PT9. As used herein, the term “PT9” refers to an antibody that binds to tau an epitope between amino acids 163 and 174 and an epitope between amino acids 219 and 226 of tau protein, has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 8, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 9. More specifically, PT9 antibody has an immunoglobulin heavy chain comprising the polypeptide sequence of SEQ ID NO: 10 and an immunoglobulin light chain comprises the polypeptide sequence of SEQ ID NO: 11.
[0064] As used herein, the term “subject” refers to an animal, and preferably a mammal. According to particular embodiments, the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, mouse, rat, rabbit, guinea pig, marmoset or mouse) or a primate (e.g., a monkey, chimpanzee, or human). In particular embodiments, the subject is a human.
[0065] As used herein a “tauopathy” encompasses any neurodegenerative disease that involves the pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplary tauopathies are frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corti cobasal degeneration, Pick’s disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy, such as dementia pugulistica (boxing disease) (Morris et al., Neuron, 70:410-26, 2011).
[0066] As used herein, the term “amyloidogenic disease” includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils. Exemplary amyloidogenic diseases include, but are not limited to systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson’s disease, Huntington’s disease, fronto-temporal dementia, and the prion- related transmissible spongiform encephalopathies (kuru and Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep and cattle, respectively). Different amyloidogenic diseases are defined or characterized by the nature of the polypeptide component of the fibrils deposited. For example, in subjects or patients having Alzheimer's disease, P-amyloid protein (e.g., wild-type, variant, or truncated P-amyloid protein) is the characterizing polypeptide component of the amyloid deposit. Accordingly, Alzheimer's disease is an example of a “disease characterized by deposits of A0” or a “disease associated with deposits of A0”, e.g., in the brain of a subject or patient. The terms “0-amyloid protein,” “[3-amyloid peptide,” “P-amyloid,” “AP” and “Ap peptide” are used interchangeably herein.
[0067] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.
[0068] As used herein, the terms “increase” and “decrease” refer to differences in the quantity of a particular biomarker in a sample as compared to a control or reference level. For example, the quantity of particular peptide, may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level. In one embodiment, an “increase of a level” or “decrease of a level” may be a difference between the level of biomarker present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more. In one embodiment, an “increase of a level” or “decrease of a level” may be a statistically significant difference between the level of the biomarker present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group. The reference or control can be, for example, a sample from a healthy individual, or a sample taken from the same individual at an earlier time point, such as a time point prior to administration of a therapeutic or an earlier time point during a therapeutic regimen.
[0069] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
[0070] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
[0071] As used herein the term “modulating, ameliorating, or treating” includes prophylaxis of a physical and/or mental condition or amelioration or elimination of the developed physical and/or mental condition once it has been established or alleviation of the characteristic symptoms of such condition.
[0072] The present application provides assays and methods for detecting singly- or multiply - ( -GlcN Acylated tau peptides in a biologic sample. The sample used in assays and methods of the present application may be a brain homogenate or a cerebrospinal fluid (CSF) sample. The biologic sample can be from a mammal, such as a mouse, non-human primate, or human. Preferably, the biologic sample is a CSF sample from a human. The assays and methods of the present application are directed to measurement of O-GlcNAcylated tau peptides in biologic samples by detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in the biologic sample. In some embodiments, the assays and methods of the present application detect O-GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208 or any combination thereof are detected. For example, the assays and methods detect (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208. In other embodiments, the assays and methods of the present application detect (9-GlcN Acylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, (iv) residue 400 or any combination are detected. In one example, the assays and methods detect O- GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400. The amount of O-GlcNAcylated tau peptides may be quantified for use in various diagnostic purposes, monitoring the level of O-GlcNAc hydrolase (OGA) inhibition, monitoring activity of an O-GlcNAc hydrolase (OGA) inhibitor in a subject, monitoring the effectiveness of a treatment such as an OGA inhibitor, identifying a subject suitable for treatment with an O-GlcNAc hydrolase inhibitor, identifying a subject suitable for a treatment for tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), pre-screening subjects for PET imaging or other diagnostic tests for detection tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), identification of subjects for enrollment in clinical trials for an O-GlcNAc hydrolase inhibitor, identification of subjects for enrollment in clinical trials relating to tauopathy or amyloidogenic disease (c.g, Alzheimer’s disease), etc.
[0073] It is believed that the concentration of tau in human CSF is three orders of magnitude lower than in the human brain. Therefore, the assays and methods of the present application preferably include a step for isolating and/or concentration the tau protein from the biologic sample (e.g., human CSF) by immunoprecipitation before analysis of the sample to detect any O- GlcNAcylation. The tau protein may be immunoprecipitated using an immunoprecipitation antibody which binds to tau. Preferably, the immunoprecipitation antibody selectively binds to human tau protein and is attached to or becomes attached to a solid phase during the process so that the immunoprecipitation antibody immobilizes tau protein in the sample to the solid phase. The solid phase may be magnetic beads, paramagnetic beads, agarose beads, or streptavidin beads. Preferably, the solid phase is magnetic beads. Supernatant from the sample may be removed and thereby isolating and/or concentrating tau protein in the remainder of the sample. [0074] In one embodiment, the immunoprecipitation antibody is a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and/or amino acids 219 and 226 of tau protein. In another embodiment, the immunoprecipitation antibody is a monoclonal antibody that binds to an epitope between amino acids 163 and 174 and an epitope between amino acids 219 and 226 of tau protein. In one exemplary embodiment, the immunoprecipitation antibody is a monoclonal antibody comprising immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively. In another exemplary embodiment, the immunoprecipitation antibody is a monoclonal antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 8, and a light chain variable region amino acid sequence of SEQ ID NO: 9. More particular, the immunoprecipitation antibody is PT9.
[0075] In one exemplary embodiment, the isolated/ concentrated tau protein is digested with at least one enzyme before analyzing the digested peptides by liquid chromatography mass spectrometry (LC-MS) to detecting O-GlcN Acylation in the tau protein. The isolated/ concentrated tau protein may be digested with trypsin, asp-N, and/or glu-C, to produce digested peptides. For example, the isolated tau protein may be divided into five (5) aliquots, which each aliquot digested under the following conditions: (1) digest with trypsin; (2) digest with asp-N; (3) digest with glu- C; (4) digest with trypsin followed by asp-N; and (5) digest with trypsin followed by glu-C. The digested peptides are then subsequently analyzed by LC-MS, such as, for example, ultra-high performance liquid chromatography- MS/MS (UHPLC-MSMS), liquid chromatography - tandem mass spectrometry (LC -MS/MS), nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS) or nanoflow liquid chromatography - high resolution mass spectrometry (nLC- HRMS), to determine amounts of O-GlcN Acylated tau peptides in the biologic sample. The LC- MS may analyze the digested peptides to determine a total amount of O-GlcN Acylated tau peptides in the sample. The LC-MS may also analyze the digested peptide to determine an amount of S184/S185 O-GlcN Acylated tau peptides, an amount of SI 91 O-GlcN Acylated tau peptides, an amount of S208 ( -GlcN Acylated tau peptides, an amount of S400 O-GlcN Acylated tau peptides, or any combination thereof.
[0076] S184/S185 O-GlcN Acylated tau peptides may be detected by LC-MS by detecting presence of a polypeptide having a sequence of SEQ ID NO: 16 or 17. SI 91 O-GlcN Acylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 18. S208 O-GlcNAcylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 19. S400 O-GlcN Acylated tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 20.
[0077] S 184/S 185 O-GlcN Acylated tau peptides may be detected by LC-MS by detecting ion transition from 600.3 m/z to 1001.5 m/z, 600.3 m/z to 996.5 m/z, or from 600.3 m/z to 798.4 m/z. S191 (9-GlcN Acylated tau peptides may be detected by detecting ion transition from 538.9 m/z to 959.5 m/z, or 538.9 m/z to 756.4 m/z. S208 (9-GlcN Acylated tau peptides may be detected by detecting ion transition from 798.9 m/z to 619.3 m/z, 798.9 m/z to 874.4 m/z, or from 798.9 m/z to 1115.5 m/z. S400 O-GlcN Acylated tau peptides may be detected by detecting ion transition from 652.8 m/z to 1101.4 m/z.
[0078] In an alternative embodiment, an assay or method 100 for detecting O-GlcNAc-tau peptides from a biologic sample from a human subject is provided, as shown in Fig. 1. The method 100 comprises a first step 102 for isolating and/or concentration the tau protein from the biologic sample by immunoprecipitation before analysis of the sample to detect any O-GlcNAcylation in the same manner as described above.
[0079] In step 104, the O-GlcNAc sites of the isolated/concentrated tau protein are labeled with biotin to produce biotinylated O-GlcNAc peptides. In one embodiment, a N- azidoacetylgalactosamine (GalNAz) group is attached to the O-GlcNAc sites of the isolated/concentrated tau protein by reacting the isolated/concentrated tau with UDP-N- azidoacetylgalactosamine (UDP-GalNAz) and Gal-Tl (Y289L). The GalNAzylated O-GlcNAc tau protein is subsequently labelled with biotin, wherein the biotin is attached to GalNAz.
[0080] In step 106, the GalNAzylated and biotinylated O-GlcNAc tau protein is digested with trypsin, asp-N, and/or glu-C, to produce digested peptides. For example, the isolated tau protein may be divided into five (5) aliquots, which each aliquot digested under the following conditions: (1) digest with trypsin; (2) digest with asp-N; (3) digest with glu-C; (4) digest with trypsin followed by asp-N; and (5) digest with trypsin followed by glu-C.
[0081] In step 108, the digested biotinylated peptides are immobilized to a solid support. For example, the digested biotinylated peptides are bound to streptavidin beads, preferably magnetic streptavidin beads, to immobilize the digested biotinylated peptides to the beads. Supernatant from the sample may be removed and thereby isolating and/or concentrating the digested biotinylated peptides.
[0082] In step 110, the immobilized and digested biotinylated peptides are reacted with Na2SO3 in a P-elimination - Michael addition reaction to release sulfited peptides from the solid support. The P-elimination - Michael addition reaction substitutes the GalNAzylated and biotinylated O-GlcNAc site with a sulfite group.
[0083] In step 112, the sulfited peptides are analyzed using LC-MS to detect sulfite modification in at least one of residue 184, 185, 191, 208, and 400 of the tau protein. The amount of sulfite modifications detected correspond to an amount of an amount of (9-GlcN Acylated tau peptides in the biologic sample. The LC-MS may analyze the digested peptides to determine a total amount of sulfite modified tau peptides in the sample. The LC-MS may also analyze the digested peptide to determine an amount of S184/S185 sulfite modified tau peptides, an amount of SI 91 sulfite modified tau peptides, an amount of S208 sulfite modified tau peptides, an amount of S400 sulfite modified tau peptides, or any combination thereof.
[0084] S184/S185 sulfite modified tau peptides may be detected by LC-MS by detecting presence of a polypeptide having a sequence of SEQ ID NO: 21 or 22. SI 91 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 23. S208 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 24. S400 sulfite modified tau peptides may be detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 25.
[0085] In one embodiment, the assays and methods of the present application measure O- GlcN Acylated tau peptides from a CSF sample from a human subject. OGA inhibition is determined to be present in the brain of the subject when the amount of O-GlcN Acylated tau peptides is above a predetermined threshold value. In another embodiment, the assays and methods of the present application measure S184/S185 O-GlcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof from a CSF sample from a human subject and the presence of OGA inhibition is determined when an amount of S 184/S 185 O-GlcN Acylated tau peptides, S 191 O-GlcNAcylated tau peptides, S208 (9-GlcN Acylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof is above a predetermined threshold value. [0086] The predetermined threshold values may be any suitable threshold value, e. g. , a suitable threshold value for distinguishing those subjects who have a decreased level of OGA inhibition as compared to those subjects who have increased level of OGA inhibition or who are healthy. The predetermined threshold values may be determined as a total O-GlcN Acylated tau peptide concentration or a concentration of S184/S185 O-GlcN Acylated tau peptides, S191 O- GlcNAcylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof for differentiating those patients that have decreased levels of OGA inhibition and those who have increased levels. Subjects identified as having a decreased level of OGA inhibition may be directed to obtain further clinical tests, such as, for example, PET imaging or other diagnostic tests, to further assess brain pathologies of these subjects. In another embodiment, subjects identified as having a decreased level of OGA inhibition may be administered an O-GlcNAc hydrolase inhibitor. Exemplary O-GlcNAc hydrolase inhibitors are described in Bartolome-Nebreda, et al., “O-GlcNAcase inhibitors as potential therapeutics for the treatment of Alzheimer’s disease and related taupathies: analysis of the patent literature,” Expert Opinion on Therapeutic Patents, Vol. 31, No. 12, 1117-1154 (2021), which is incorporated in its entirety by reference herein.
[0087] In one example, the O-GlcNAc hydrolase inhibitor is Thiamet G having the structure of Formula I:
Figure imgf000024_0001
(Formula I) or a derivative thereof. In another example, the O-GlcNAc hydrolase inhibitor is (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2- d]thiazole-6,7-diol (MK-8719), or N-[4-fluoro-5-[[2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]piperidin-l-yl]methyl]-l,3-thiazol-2-yl]acetamide (LY-3372689).
[0088] In another embodiment, the assays and methods of the present application measure O- GlcNAcylated tau peptides from a CSF sample from a human subject and subsequently determines that the subject is need of ( -GlcN Ac hydrolase inhibition. Alternatively, the assays and methods of the present application measure ( -GlcN Acylated tau peptides from a CSF sample from a human subject and subsequently determines that the subject is in need of O-GlcNAc hydrolase inhibition when an amount of S 184/S 185 G-GIcN Acylated tau peptides, S 191 G-GIcN Acylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 O-GlcNAcylated tau peptide, or any combination thereof is below a predetermined threshold value. The predetermined threshold values may be any suitable threshold value, e.g., the predetermined threshold values may be determined as a total O- GlcNAcylated tau peptide concentration or a concentration of SI 84/S 185 G-GIcN Acylated tau peptides, SI 91 G-GIcN Acylated tau peptides, S208 G-GIcN Acylated tau peptides, S400 O- GlcNAcylated tau peptide, or any combination thereof for differentiating those patients that are healthy and those patients that have an G-GIcN Acylation modulated disease.
[0089] In some embodiments, the predetermined threshold value may correspond to a baseline value or a value that is significantly higher than the baseline value. As used herein, “significantly higher” refers to a higher value that is statistically significant, not due to chance alone, which has a p-value of 0.05 or less. “Significantly higher” can be at least about 1%, 2%, 5%, or 10% higher than that found in healthy subjects, at a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc. The baseline value may correspond to a mean level in a population of healthy individuals. The baseline value may also correspond to a mean value of previous levels determined in the same subject.
[0090] In another embodiment, a method of the present application comprises (i) detecting O- GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcN Acylated tau peptides in a biologic sample obtained from the subject; and (ii) determining the effectiveness of an O-GlcNAc hydrolase inhibitor administered to the subject based a total amount of G-GIcN Acylated tau peptide or an amount of S 184/S 185 G-GIcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 O- GlcNAcylated tau peptide, or any combination thereof measured.
[0091] In yet another embodiment, effectiveness of a treatment, e.g., an O-GlcNAc hydrolase inhibitor, administered to the subject is determined by monitoring a total amount of O- GlcNAcylated tau peptide or an amount of SI 84/S 185 G-GIcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 O-GlcNAcylated tau peptide, or any combination thereof, before, during, and/or after administration of the treatment. An increase in values relative to baseline signals an increase in G-GIcN Ac hydrolase inhibition. [0092] In some embodiments, the treatment can be treatment for a neurodegenerative disease, such as a tauopathy and/or amyloidogenic disease. The tauopathy and/or amyloidogenic disease may be selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, nonGuamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugulistica (boxing disease). Preferably, the tauopathy and/or amyloidogenic disease is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease), FTDP-17 or progressive supranuclear palsy. Most preferably, the tauopathy and/or amyloidogenic disease is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease).
[0093] In another embodiment, a method of the present application comprises (i) detecting O- GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcN Acylated tau peptides in a biologic sample obtained from the subject; and (ii) determining the effectiveness of treatment with an O-GlcNAc hydrolase inhibitor administered to the subject based on a total amount of O-GlcN Acylated tau peptide or an amount of S 184/S 185 (9-GlcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O-GlcN Acylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof measured.
[0094] In yet another embodiment, effectiveness of treatment with an O-GlcNAc hydrolase inhibitor administered to the subject is determined by monitoring a total amount of O- GlcNAcylated tau peptide or an amount of SI 84/S 185 O-GlcN Acylated tau peptides, SI 91 O- GlcNAcylated tau peptides, S208 O-GlcNAcylated tau peptides, S400 (9-GlcN Acylated tau peptide, or any combination thereof, before, during, and/or after administration of the treatment. An increase in values relative to baseline signals an increase in an O-GlcNAc hydrolase inhibition. [0095] According to one embodiment, a method of the present application comprises (i) detecting (9-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcNAcylated tau peptides in a biologic sample obtained from the subject; and (ii) determining whether or not the subject is suitable for treatment with an O-GlcNAc hydrolase inhibitor based on a total amount of O-GlcN Acylated tau peptide or an amount of S184/S185 O-GlcN Acylated tau peptides, SI 91 O-GlcN Acylated tau peptides, S208 O- GlcNAcylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof measured. According to a particular aspect, it is determined that a subject is suitable for treatment with an O-GlcNAc hydrolase inhibitor if the total amount of O-GlcNAcylated tau peptide or the amount of S184/S 185 G-GIcN Acylated tau peptides, SI 91 (9-GlcN Acylated tau peptides, S208 O- GlcNAcylated tau peptides, S400 O-GlcN Acylated tau peptide, or any combination thereof measured is lower than a pre-determined threshold value.
[0096] In some embodiments, the method further comprises a step of administering an OGA inhibitor to a subject determined to be in need of OGA inhibition or to a subject determined to be suitable for treatment with an OGA inhibitor.
EXAMPLES
[0097] The following examples are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims.
Example 1: IP-LC-MSMS of tau O-GlcNAcylated at S400 in Mouse Brain Homogenates
[0098] Example 1 provides an exemplary method for determining whether tau O- GlcNAcylated at S400 could be captured and detected with IP-LC-MSMS methodology using a total tau antibody. A showing that IP-LC-MSMS methodology is capable of detecting tau O- GlcNAcylated at S400 demonstrates that the IP-LC-MSMS methodology can also be applied to identifying other tau (9-GlcN Acylation sites in mouse brain homogenates (BH).
Materials
[0099] Ammonium bicarbonate, formic acid (LA) 98-100 %, methanol (for spectroscopy), chloroform, Tween20, ammonia solution 25 % (Suprapur), sodium hydroxide (NaOH), 1 M HEPES and bovine serum albumin (BSA) Cohn fraction V were supplied by Merck. MilliQ water with a resistivity of 18.2 MQ.cm at 25 °C was generated with a Millipore™-purification system. Trifluoroacetic acid (TFA) and sodium sulfite (Na2SCh) were purchased from VWR. Acetonitrile (ULC-MS) was bought from Actu-All Chemicals. Recombinant tau-441 was acquired from Promise Proteomics. Synthetic peptide H-SPVV-(O-GlcNAc)S-GDTSPR-OH (SEQ ID NO: 26) was supplied by Tebu Bio, H-SPWSGDTSPR-OH (SEQ ID NO: 27) by GenScript, and peptides H-TPSLPTPPTR-OH (SEQ ID NO: 28), H-TPSLP(pT)PPTR-OH (SEQ ID NO: 29), H- SR(pT)PSLP(pT)PPTREPK-OH (SEQ ID NO: 30), H-TPSLPTPPTR*-OH (SEQ ID NO: 31) and H-TPSLP(pT)PPTR*-OH ((SEQ ID NO: 32) (*: Arginine labelled with 13C and 15N) were purchased from New England Peptide. Octyl P-D-glucopyranoside and Dulbecco's phosphate buffered saline (DPBS) were supplied by Thermo Scientific. Antibody PT9 was produced. Trypsin Gold, asp-N sequence grade and glu-C sequence grade were obtained from Promega.
Mice and treatments
[00100] One month-old animals (males and females) were treated for 20 weeks with Thiamet G, an O-GlcNAc hydrolase inhibitor, in drinking water (tap water); high dose groups (HIGH1 and HIGH2) corresponding to 2.5 mg/mL (500 mg/kg/day target doses based on fluid consumption) treated groups from 2 independent studies (Study 1 and Study2), low dose group (L0W2) corresponding to a 0.139 mg/ml (28 mg/kg/day target dose) treated group from a single study (Study 2) and control group (CONTR1) corresponding to littermates on regular drinking water without treatment. All littermates were randomly assigned to treatment groups. Five tau knock-out (KO) mice brain from 16-20-weeks old animals were homogenized and used as tau negative controls.
Brain homogenates preparation
[00101] Brains were rapidly harvested, immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. Brains were homogenized in buffer H in a 1:6 ratio (weight (mg):buffer volume (pL)), using Lysing matrix D tubes (MP Biomedicals #6913-500) in a Fastprep-24 Homogenizer (MP Biomedicals, 116005500 6.0 m/s, time 20 s). Homogenates were then centrifuged at 20,000 g, at room temperature, for 40 minutes to remove cell debris. Individual supernatants were collected, and equal amounts of individual samples per treatment group were pooled together to create pools (pool HIGH1 (n=23), pool HIGH2 (n=27), pool L0W2 (n=27)). Pools were stored at -80°C until analysis. For quantification of S400 O-GlcNAc purposes, brain homogenates were subsequently diluted in buffer H at a 1 :30 ratio. Preparation of standard dilutions
[00102] All standard dilutions were prepared in low bind Eppendorf tubes. Non-labelled and stable isotopically labelled (SIL) peptide standard solutions: individual stock solutions of 200 pM were prepared in water:acetonitrile:acetic acid (89: 10:1). The stock solutions were aliquoted and stored at -80°C. Standard working dilutions were freshly prepared for each batch of samples in 90:10:0.1 water: acetonitrile: formic acid.
Tau extraction from mouse brain homogenates
Immunoprecipitation
[00103] A method for concentrating tau and O-GlcNAc-tau protein from a biologic sample using immunoprecipitation (IP) was used to obtain tau extract from mice brain homogenates as described further below. Per IP reaction, 93.5 pL of Dynabeads protein G (Thermo Fisher Scientific) corresponding to 2.8 mg beads was washed two times in LoBind Eppendorf tubes (Eppendorf) with 125 pL 0.01% Tween20 in PBS. The tubes were placed in a DynaMag™-2 Magnet (Thermo Fisher Scientific) and the supernatant was discarded. 16.6 pL PBS + 0.1% Tween20, lOOnM of the PT9 antibody (final concentration in incubate) and 150 pL sample (either biologic sample (e.g., brain homogenate) 30-fold diluted in buffer H, or buffer H (method blank)) were added to the beads. Buffer H (high Salt buffer) consisted of 10 mM Tris (Thermo Fisher #15567-027), 800 mM NaCl (Thermo Fisher #24740-011), 1 mMEDTA (Thermo Fisher #15575- 020), 10 % sucrose (Sigma #S9378), pH 7.4, filtered on 0.22 pm and supplemented with protease cocktail (Roche Complete mini EDTA free #11836170001) and phosphatase cocktail inhibitors (Roche Phostop #04906845001) as recommended by the manufacturer. The samples were mixed and incubated overnight at 4°C while rotating on a hula mixer (Thermo Fisher Scientific). The supernatant was collected as immunodepleted fraction. 150 pL (50 mM ammonium bicarbonate (pH 8) + 10% of 0.1% Tween20 in PBS) was added to the beads and subsequently vortex mixed and spun (Minispin Plus centrifuge, Eppendorf). The supernatant was collected as wash and the beads were resuspended in 150 pL of 50 mM ammonium bicarbonate, pH 8. The samples were stored on melting ice while preparing reagents for trypsinization: trypsinization was started on the day IP was finished.
Trypsinization [00104] 15 JJ.L of acetonitrile was added to the beads + 150 pL of 50 mM ammonium bicarbonate, vortex mixed for 5 s and spun down for 15 s with a Minispin Plus centrifuge. 25 pL of 0.05 mg trypsin mL'1 in 50 mM acetic acid was added and the samples were subsequently incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific) for on-bead digestion. Afterwards, the digestion was quenched by adding 15 pL of formic acid and briefly vortex mixing.
[00105] Subsequently, 40 pL of SIL working solution containing 0.5 nM H-TPSLPTPPTR*- OH (SEQ ID NO: 31) and H-TPSLP(pT)PPTR*-OH (SEQ ID NO: 32) in 90:10:0.1 water: acetonitrile: formic acid was added. The samples were vortex mixed and centrifuged at 20,000*g for 10 min (Heraeus Megafuge 8R centrifuge, Thermo Fisher Scientific) and the supernatant was transferred to micronic tubes (Micronic). The micronics were sealed in a 96 well plate with sealing mat and stored at 4°C until analysis.
[00106] A solution containing approximately 3 ng/mL recombinant tau 2N4R (recombinant tau- 441 from Promise Proteomics) in 50 mM ammonium bicarbonate was digested in parallel with the biologic samples as positive control for trypsinization. 150 pL 50 mM ammonium bicarbonate was digested under the same conditions as digestion method blank. Next to that, aliquots of 90 pL were taken from each solution (the immunoprecipitated and digested samples, digested recombinant tau 2N4R, and buffer H), and spiked with 10 pL of a 1 nM standard mix in 90:10:0.1 water: acetonitrile: formic acid containing the above non-labelled surrogate peptides to check ionization suppression effects.
LC-MS analysis
UHPLC-MSMS analysis of brain homogenate extracts for the detection of tau S400 O- GlcNAcylation
[00107] Ultra-high performance liquid chromatography- MS/MS (UHPLC-MSMS) analyses were performed on an ultra-high performance liquid chromatograph from Shimadzu consisting of 2 Nexera LC30AD liquid pumps set up to provide binary solvent gradients, a SIL-AC30 autosampler, a CTO-20AC column oven, a communications bus module (CBM-20A) and a sample Rack Changer II, hyphenated via a Turbo-Ionspray™ Interface (Sciex) to a 6500 triple quadrupole mass spectrometer (Sciex). Analyst versions 1.6.3 and 1.7 (Sciex) was used as instrument control and data processing software. 50 pL of digest was injected on a YMC-Triart Cl 8 column, 2.0 mm x 50 mm, 1.9 pm (YMC) and thermostatically (60 °C) eluted.
[00108] The mobile phase (MP) solvents consisted of 100:1 water + 0.05 % ammonia: acetonitrile (v:v) (A) and acetonitrile + 0.05 % ammonia (v:v) (B), and the gradient was set as follows (min/A%): 0.0/100, 5.0/70, 5.1/2, 6.6/2, 6.7/100, 10/100. The flow rate was set at 0.5 mL/min. The peptides were ionized with electrospray ionisation (ESI) in positive ion mode. The ionspray voltage was set to 5500 V, temperature to 450°C, declustering potential to 86 V and entrance potential to 10 V. Ion source gas 1, gas 2 and curtain gas were set to 50, 40 and 30, respectively. CAD gas was set to 6. [00109] The selected MS transitions used for multiple reaction monitoring of the target peptides are provided in Table 1, which provides MRM-transitions used in UHPLC-MSMS experiments for the detection of tau S400 O-GlcNAcylation in mouse brain homogenate samples. Transitions were divided into two periods: period 1 from 0 to 2.6 min. and period 2 from 2.6 to 10 min. Data processing was performed with Analyst 1.7 or Sciex OS 1.6 (Sciex). Table 1.
Figure imgf000031_0001
^Stable isotopically labelled standard
Results and Discussion Identification of S400 O-GlcNAcylation in mouse brain homogenates
[00110] Brain samples from two independent animal studies (Studyl and Study2) investigating the effect of Thiamet G treatment were analyzed. In both studies, transgenic P301S mice overexpressing human tau were administered Thiamet G for 5 months via drinking water. P301S mice kept without Thiamet G treatment (vehicle) were used as control. P301S mutation is found in familial frontotemporal dementias with tau aggregation.
[00111] Pools of cortex from treatment and control groups, and additionally also tau knock-out (KO) mice were prepared separately by homogenization in buffer H. The homogenates (and buffer H as method blank) were immunoprecipitated with antibody PT9, a total tau antibody with two epitopes in the proline rich region (163 - 174 and 219 - 226, numbering based on tau isoform 2N4R). Immunoprecipitation was followed by on-bead digestion with trypsin to obtain surrogate peptides. Extracts were analyzed with UHPLC-MSMS.
[00112] Solvent dilutions of surrogate peptide standards SPW(S400)GDTSPR (SEQ ID NO: 13) and SPW-((G-GlcNAc)S400)-GDTSPR (SEQ ID NO: 20) were used for identification of non-modified tau and tau (9-GlcN Acylated at S400 in the samples. Although during tuning of the MRM-transitions for SPW(S400)GDTSPR (SEQ ID NO: 15), the presence of diagnostic ions for the (9-GlcN Acylation were observed (m!z 126; 138; 144; 168; 186 and 204), no sensitive diagnostic product ions to localize O-GlcNAcylation at S400 were observed: non-diagnostic MRM-transitions were thus used.
[00113] A second set of surrogate peptides, namely TPSLP(T217)PPTR (SEQ ID NO: 36), TPSLP(pT217)PPTR (SEQ ID NO: 34), and SR(pT)PSLP(pT217)PPTREPK (SEQ ID NO: 35), representing tau either non-modified or phosphorylated at or around T217, was used to monitor the influence of Thiamet G treatment on tau T217 phosphorylation. Tryptic peptide concentrations in the sample extracts were calculated using standard dilutions in solvent (no matrix matching). As S400 is remote from PT9 epitopes and not located next to trypsin cleavage sites, it is expected that O-GlcNAcylation at S400 will not influence IP and trypsinization. It was therefore assumed that sample preparation did not bias the calculation of the relative abundance of ‘tau O- GlcNAcylated at S400’ versus ‘non-modified tau + tau O-GlcN Acylated at S400’. Spiking peptide standards to the final extracts of tauKO samples and IP method blank (immunoprecipitated buffer H) revealed a reduction in peak areas in comparison with solvent standards. The matrix ionization suppression was similar in tauKO samples and IP method blanks (immunoprecipitated buffer H). [00114] The relative concentrations of the monitored tau post-translational modifications (PTMs) in the samples, calculated as ‘concentration surrogate peptide with PTM * 100 / (concentration surrogate peptide with PTM + concentration non- modified surrogate peptide)’, are presented in Table 2. Table 2 shows relative concentrations of monitored post-translational modifications of tau (S400 O-GlcNAcylation and phosphorylation in the mid-region (at and around T217)) in brain homogenates of P301S transgenic mice from 2 independent Thiamet G treatment studies (Study 1 and Study2), calculated as ‘concentration surrogate peptide with PTM * 100 / (concentration surrogate peptide with PTM + concentration non-modified surrogate peptide)’, expressed in %. The data of Table 2 are obtained with IP-LC-MSMS (semi-quantitative data), with the results being averages of duplicates.
Table 2.
Figure imgf000033_0001
[00115] No surrogate peptides of tau were detected in tauKO and buffer H samples, indicating that the used IP-LC-MSMS methodology is selective for the measurement of the surrogate peptides of interest. The concentrations of surrogate peptides representing non-modified tau (SPWSGDTSPR (SEQ ID NO: 15) and TPSLPTPPTR (SEQ ID NO: 36)) and tau phosphorylated in the mid region (TPSLP(pT)PPTR (SEQ ID NO: 34) and SR(pT)PSLP(pT)PPTREPK (SEQ ID NO: 35)) remained constant over the different P301S BH samples analyzed, with concentration fold-changes ranging between 0.82 - 1.1 for Thiamet G treated samples in comparison with vehicles: Thiamet G treatment did not affect the degree of phosphorylation around T217 significantly (as can also be observed from the relative (%modified tau) amounts of phosphorylated tau in Table 2). [00116] A similar increase in S400 O-GlcNAcylation is observed for the high dosing group (Table 2), with concentration fold-changes of 11 in Study 1 and 13 in Study2 with respect to vehicle samples. Also for the low dosing group an increase is observed, but less pronounced than with the high dose (concentration fold-change: 4). These data thus show that a change in O-GlcN Acylation at S400 of tau can be monitored in mouse BH with the applied IP-LC-MSMS methodology. Moreover, as the applied immunoprecipitation protocol makes use of a total tau Ab, the obtained BH extracts could also contain tau O-GlcNAcylated at other sites.
Example 2: IP-LC-MSMS of U-GIcN Acylation sites in Recombinant Human Tau
[00117] Example 2 describes an exemplary method for identifying O-GlcNAc-sites from O- GlcNAc rec htau produced in the manner described below. Identification of ( -GlcN Acylated tau peptides at relatively high concentrations present in an O-GlcNAc rec htau digest would allow gathering of high-quality LC-MSMS data, to be used as reference for identification of low concentration (9-GlcN Ac-peptides in mouse BH extracts in Example 4.
[00118] Unless indicated otherwise below, the materials used in Example 2 are the same as described above or Example 1.
Production of O-GlcNAcylated recombinant human tau (2N4R isoform)
[00119] The O-GlcNAcylated tau expression and purification was performed as described below.
[00120] Expression construct generation'. (1) the DNA sequence encoding full length human OGT protein (SEQ ID NO: 37) (Uniprot 015294-3) was synthesized and codon optimized for expression in E. coli and subcloned in frame with the MBP sequence of the pMAL-c5X vector (NEB); and (2) the DNA sequence encoding human MAPT-tau 2N4R protein of SEQ ID NO: 1 (Uniprot Pl 0636-8) was synthesized and codon optimized for expression in /A coli and subcloned in frame with the N-terminal 6xHis-tag sequence of the pET28a(+) vector (NEB), a stop codon was included to prevent the C-terminal His tag readthrough.
[00121] Tau and OGT Co-expression. E. coli strain T7-Express (NEB) was transformed with the human MAPT-tau 2N4R and OGT encoding plasmids and double transformants selected on LB-Agar plates with 50 pg/mL carbenicillin and 25 pg/mL kanamycin. Next morning, colonies were used to set up expression cultures in 2.5 L Ultra Yield flasks (Thomson) containing 1 L LB medium, 50 .g carbenicillin and 25 pg/mL kanamycin, at 37°C and 300 RPM shaking. Once OD600nm reached 0.5, cultures were cooled to 22°C and expression induced with 0.5 mM IPTG overnight. In some cases, the LB medium was supplemented with 1 % glucose during expression. Next morning cells were harvested by centrifugation at 6000 RPM.
[00122] Purification', all chromatography media and the FPLC system were from Cytiva. All purification steps described in this paragraph were performed at 4°C. The E. coli cell pellet from centrifugation was resuspended in 10 volumes of buffer A (50 mM Na2HPO4, 0.5 M NaCl, 5 mM P-mercaptoethanol and protease inhibitor mix (Roche), pH 7.8) supplemented with 15 mM Imidazole and lysed by sonication. Insoluble material was removed by centrifugation for 30 min at 25000xg. His-tagged tau protein was captured on Nickel sepharose 6-FF beads (0.75 mL / L Culture) in batch binding mode by gentle mixing for 1.5 hours. The beads were captured and washed by three rounds of centrifugation and re-suspension with 10 volumes buffer A supplemented with 30 mM Imidazole. The beads were packed on an empty C-10 column connected to a AKTA Pure FPLC and washed to stable A280nm baseline with the same buffer. Tau protein was eluted with buffer A supplemented with 500 mM Imidazole and peak fractions analysed by SDS-PAGE. The purest fractions were pooled, concentrated, and loaded immediately on a Superdex 200 pg 16/600 SEC column, pre-equilibrated with SEC buffer (25 mM Tris, 150 mM NaCl, 1 mM DTT). The column was run at 1.5 mL/min with SEC buffer, UV monitored at 280 nm and fractions collected. Peak fractions were analyzed by SDS-PAGE, purest fractions pooled, flash frozen and stored at -80°C as aliquots.
Digestion of O-GlcNAcylated recombinant human tau with multiple enzymes
[00123] (9-GlcN Ac rec htau was digested with multiple enzymes in parallel (trypsin, asp-N and glu-C): digestion with proteases asp-N and glu-C could provide complementary information with regard to trypsin, because of their ability to cleave at different amino acids (N-terminal of D, and C-terminal of E and D, respectively), thus resulting in different peptides. Digestion with two enzymes was performed with trypsin before asp-N, and trypsin before glu-C. Specifically, O- GlcNAcylated recombinant human tau (O-GlcNAc rec htau), produced in the manner described above, was digested with different enzymes, rendering 5 digestion conditions: (1) trypsin only, (2) asp-N only, (3) glu-C only, (4) trypsin followed by asp-N, and (5) trypsin followed by glu-C. [00124] For each digestion condition, 20 pL of 190 pg/mL O-GlcNAc rec htau was aliquoted to a low bind Eppendorf tube, 180 pL of 50 mM ammonium bicarbonate and 20 pL acetonitrile were added, followed by vortex mixing for 5 s and spinning down for 15 s. For digestion conditions 1, 4 and 5, 19 pL of 0.01 mg trypsin ml 1 in 50 mM acetic acid was added (freshly prepared). For digestion condition 2, 19 pL of 0.004 mg asp-N m '1 in 50 mM ammonium bicarbonate was added (freshly prepared). For digestion condition 3, 19 pL of 0.01 mg glu-C mL'1 in 50 mM ammonium bicarbonate was added (freshly prepared). Remaining unused dilutions of the digestive enzymes were stored at -20°C until further use.
[00125] The O-GlcNAc rec htau samples were incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific) for digestion. For conditions 1, 2 and 3, the digestion was quenched afterwards by adding 30 pL of formic acid. For digestion condition 4, 19 pL of 0.004 mg asp-N mL’1 in 50 mM ammonium bicarbonate, was added and the mixtures were again incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific). For digestion condition 5, 19 pL of 0.01 mg glu-C mL’1 in 50 mM ammonium bicarbonate, was added and the mixtures were again incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific). The digestion was quenched by adding 30 pL of formic acid.
[00126] A summary of the digestion conditions are provided below in Table 3.
Table 3.
Figure imgf000036_0001
[00127] All digests were vortex mixed and centrifuged at 20,000 g for 10 min (Heraeus Megafuge 8R centrifuge, Thermo Fisher Scientific) immediately after quenching and the supernatant was transferred to injection vials (BGB ANALYTIK), capped and stored at -20°C until analysis.
LCMS Analysis nLC-HRMS analysis of O-GlcNAcylated recombinant human tau digests
[00128] Chromatographic analysis of O-GlcN Acylated recombinant human tau digests was performed on a Thermo Fisher Scientific Ultimate 3000 RSLC nano LC System with Pro Flow technology (Thermo Fisher Scientific). 10 pL of digest was injected and loaded on an Acclaim™ PepMap™ 100 C18 trap column, 5 pm, 0.3 mm x 5 mm (Thermo Fisher Scientific): 98:2 water: acetonitrile + 0.05% TFA was pumped isocratically at 10 pL/min with a pLC loading pump. A 10-port switching valve was used to divert the eluate of the trap column to waste. After 2 min of sample loading, the valve was switched to elute the trapped analytes in backflush mode onto an EASY-Spray PepMap Cl 8 nLC column, 3 pm, 75 pm x 15 cm (Thermo Fisher Scientific). The analytes were chromatographed with a linear gradient provided at 300 nL/min by a nLC binary pump, with mobile phases A: 100% water + 0.1% FA, and B: 20:80 water: acetonitrile + 0.1% FA. Details of the chromatographic conditions are provided in Table 4.
[00129] Table 4 provides LC-gradient and valve switching timing of the nLC-HRMS method used for analysis of (9-GlcNAcylated recombinant human tau digests. Mobile phase solvents A and B are pumped by the nLC binary pump, mobile phase solvent C is provided by the pLC loading pump. Valve position ‘O’: pLC flow passes via sample loop through the trap column and finally elutes to waste (load position). Valve position ‘ 1’: nLC flow passes through trap column and analytical column to finally enter the MS system. The column ‘Parameter’ indicates valve position (0 or 1), and percentage (%) B and C solvent, respectively.
Table 4.
Figure imgf000037_0001
Figure imgf000038_0001
[00130] Both columns and the 10-port switching valve were heated to 40°C in a column thermostat. The eluant of the nLC column was transferred via an EASY-spray source (Thermo Fisher Scientific) to a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).
[00131] For data dependent MS2 (ddMS2) experiments, centroid data were recorded with electrospray ionisation mode in positive polarity. Full MS data were acquired with scan range set from m/z 375 to m/z 1,500. Resolution in full MS mode was set to 60,000 FWHM, with Automatic Gain Control (AGC) target at 3e6 and maximum injection time of 60 ms. Polysiloxane was used for lock mass correction. Resolution for ddMS2 was set to 15,000 with AGC target at le5 and maximum injection time of 50 ms (loop count 3 and topN set to 3). The isolation window of the quadrupole was set to 1.6 m/z and scan range of ddMS2 experiments was set from m/z 200 to m/z 2,000. Normalized collision energy was set to 28. Data dependent fragmentation settings: minimum AGC target: 2e3, intensity treshold: 4e4, charge-state exclusion: unassigned, 1, 7, 8, >8, exclude isotopes: on, dynamic exclusion: 5 s.
[00132] For peptide TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85 (SEQ ID NO: 16 or 17), an additional parallel reaction monitoring (PRM) experiment was set up. The doubly charged peptide TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) at m/z 600.29320 was added to an inclusion list for fragmentation. Resolution for PRM was set to 30,000 with AGC target at 2e5 and maximum injection time of 50 ms. The isolation window of the quadrupole was set to 1 m/z. Normalized collision energy was set to 28. Data were recorded in profile mode. nLC-HRMS data processing
[00133] XCalibur 3.0 (Thermo Fisher Scientific) was used for nLC-HRMS data acquisition and manual data processing. Proteome Discoverer 2.3 (Thermo Fisher Scientific) was used to create a peak list from the raw data with ‘Fixed Value PSM Validator’ node, with the value for 'Peptide Confidence At Least' set to 'Medium'. Maximum missed cleavages was set to 2, minimum peptide length to 6 AAs, precursor mass mass accuracy: 10 ppm, fragment mass mass accuracy: 0.02 Da, and selected dynamic modification: HexNAc. A FASTA-file with the protein sequence of human tau 2N4R isoform F of SEQ ID NO: 1 (Pl 0636 downloaded from uniprot.org) was used as protein database.
Results and Discussion
Determination of O-GlcNAc-sites in O-GlcNAcylated recombinant human tau
[00134] The obtained O-GlcNAc rec htau digests were analyzed with nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS). MS fragmentation spectra obtained with data dependent acquisition were used for identification of O-GlcNAc-peptides. Proteome Discoverer with node 'Fixed Value PSM Validator' was used to extract potential O- GlcNAc-peptide MS fragmentation spectra from the raw data. The MS fragmentation spectra of positive hits were manually inspected. For O-GlcNAc-peptides present in low abundance, additional parallel-reaction monitoring (PRM) experiments were carried out to obtain more sensitive and high-quality fragmentation spectra. CID predominantly resulted in y- and b-ions, often with loss of the O-GlcNAc-moiety in O-GlcNAcylated peptides. Location of the O-GlcNAc- site was therefore not always possible. MS fragmentation spectra of ( -GlcN Acylated peptides, on the other hand, resembled their non-modified counterparts well. O-GlcNAc signature product ions (m/z 126, 138, 144, 168, 186 and 204) provided confirmation of the presence of a HexNAc-moiety. Furthermore, O-GlcNAc-peptides eluted just before -or co-eluted with- their non-modified counterpart, thereby giving additional confirmation of their identity. Extracted ion chromatograms and MS fragmentation spectra of the below discussed peptide identifications are provided in Figs. 2-13.
[00135] Analysis of the digests with nLC-HRMS predominantly resulted in the detection of some high abundant C-terminal O-GlcNAcylated peptides. This data suggests that in Example 2, tau was predominantly O-GlcNAcylated in the C-terminal region. Digestion with glu-C resulted in the detection of only large C-terminal O-GlcNAc-peptides, while digestion with trypsin followed by glu-C did not render any additional O-GlcNAc-peptides with regard to the trypsin digest. The glu-C digests were therefore not further evaluated. [00136] The C-terminal tau peptide SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), previously identified in mouse brain homogenates in Example 1 , was also detected in a trypsin digest of O-GlcNAc rec htau. Fig. 2 shows extracted ion chromatograms of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) (5 ppm mass extraction window). The mass of the precursor ion non-modified peptide is at m/z 551.28038 (doubly charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 652.82006 (doubly charged). The percentage S400 O-GlcNAcylation in O-GlcNAc rec htau, calculated with surrogate peptide standards, was found to be 9.5%. MS fragmentation data were investigated: although some minor product ions containing an O-GlcNAc-moiety were detected, the site of O- GlcNAcylation could not be unambiguously assigned with this experiment.
[00137] Two isomeric HLSNVSSTGSI*O-GlcNAc peptides (peptide of SEQ ID NO: 38 having at least one O-GlcNAc site) originating from the C-terminus of tau were identified in a trypsin - asp-N digest of O-GlcNAc rec htau (with the same exact mass as SPW-(O-GlcNAc)S- GDTSPR(SEQ ID NO: 20)). Fig. 3 shows MS fragmentation spectra of peptides SPWSGDTSPR (SEQ ID NO: 15) and SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20). Diagnostic O-GlcNAc ions (indicated by solid arrows to the left side) are predominantly lost upon fragmentation. Dotted arrows to the right side indicate product ions with O-GlcNAc modification. Table 5 below identifies product ions of the SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) peptide, with the product ion annotation based on O-GlcNAcylation at S400. The product ions y7 and 78 are listed in bold to reflect those being product ions that contain O-GlcNAc.
Table 5.
Figure imgf000040_0001
Figure imgf000041_0001
[00138] Due to in-source loss of the (9-GlcN Ac-moiety, it was not possible to pinpoint the exact location of O-GlcNAc. No di-O-GlcNAc peptide was detected, suggesting that both O-GlcNAc modifications at vicinal positions are mutually exclusive. The above data on C-terminal O- GlcNAcylation show that incubation of full-length tau with OGT resulted in 3 major O-GlcNAc- sites, all found at the C-terminal residues S400, S412 and S413.
[00139] A couple of low abundant O-Glcnac peptides originating from the N-terminal side and mid-domain of tau were detected. Fig. 4 shows extracted ion chromatograms of peptides HLSNVSSTGSI (SEQ ID NO: 34) and HLSNVSSTGSI*O-GlcNAc isomers (5 ppm mass extraction window). The mass of the precursor ion non-modified peptide is at m/z 551.28038
(doubly charged) and the mass of the precursor ions O-GlcNAcylated peptides is at m/z 652.82006 (doubly charged). Fig. 5 shows MS fragmentation spectra of peptides HLSNVSSTGSI (SEQ ID NO: 34) and HLSNVSSTGSI*O-GlcNAc isomers. Diagnostic O-GlcNAc ions (indicated by solid arrows to the left side) are predominantly lost upon fragmentation. Table 6 below identifies product ions of HLSNVSSTGSI*O-GlcNAc peptide eluting at 15.53 min. Product ion annotation is based on HLSNVSSTGSI (SEQ ID NO: 34) due to in-source loss of O-GlcNAc-moiety.
Table 6.
Figure imgf000041_0002
Figure imgf000042_0001
[00140] The peptide SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) was identified based on several diagnostic product ions, locating the O-GlcNAcylation at S208 (based on 2N4R tau isoform): particularly the product ion at m/z 619.3047, originating from y4-fragmentation with retention of the O-GlcNAc-moiety, provided unambiguous confirmation. Fig. 6 shows extracted ion chromatograms of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 14) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) (5 ppm mass extraction window). The mass of the precursor ion non-modified peptide is at m/z 697.32076 (doubly charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 798.86045 (doubly charged). Fig. 7 shows MS fragmentation spectra of peptides SGYSSPGSPGTPGSR (SEQ ID NO: 14) and SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19). Diagnostic O-GlcNAc ions (indicated by solid arrows on the left) are predominantly lost upon fragmentation. Dotted arrows on the right indicate product ions with O-GlcNAc modification. Table 7 below identifies product ions of SGYSSPGSPGTPG-(O-GlcNAc)S-R (SEQ ID NO: 19) peptide. The product ions y4, y7, y8, y9, ylO, yl l and y 12 are listed in bold to reflect those being product ions that contain O-GlcNAc. The product ion originating from y4 fragmentation indicates O-GlcN Acylation at S208.
Table 7.
Figure imgf000042_0002
Figure imgf000043_0001
[00141] Another tryptic peptide TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) with one missed cleavage, corresponding to O-GlcN Acylation at SI 91, was detected: although the chromatographic peak shapes of the unmodified and ( -GlcN Acylated peptide were broad, they matched well in terms of retention and shape. Fig. 8 shows extracted ion chromatograms of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18)(5 ppm mass extraction window). The mass of the precursor ion non-modified peptide is at m/z 471.23192 (triply charged) and the precursor ion O-GlcNAcylated peptide: m/z 538.92504 (triply charged). Fig. 9 shows MS fragmentation spectra of peptides TPPSSGEPPKSGDR (SEQ ID NO: 13) and TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO:
18). Diagnostic O-GlcNAc ions (indicated by solid arrows to the left) are predominantly lost upon fragmentation. Dotted arrows to the right indicate product ions with O-GlcNAc modification. Table 8 identifies product ions of TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18) peptide. The product ions yl2 and y7 are listed in bold to reflect those being product ions that contain O- GlcNAc. The product ion originating from y7 fragmentation indicates O-GlcN Acylation at SI 91.
Table 8.
Figure imgf000044_0001
[00142] Notwithstanding that the fragmentation spectrum confirmed the peptide AA sequence, only 2 product ions were detected with retention of the O-GlcNAc-moiety. The product ion with m/z 959.4859 resulting from y7 fragmentation allowed to pinpoint S191 as O-GlcNAcylation site. [00143] Yet another tryptic peptide originating from the same part of the tau sequence, namely
TPPSSGEPPK (SEQ ID NO: 12), but not containing S 191 , was detected with O-GlcNAcylation. As for TPPSSGEPPK-(O-GlcNAc)S-GDR (SEQ ID NO: 18), broad chromatographic peak shapes were obtained for both the unmodified and O-GlcNAc peptide, matching well however in terms of shape and retention. Fig. 10 provides extracted ion chromatograms of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc representing tau O-GlcNAcylation at either S 184 or S 185 (SEQ ID NO: 16 or 17) (5 ppm mass extraction window). The mass of the precursor ion non-modified peptide is at m/z 498.75346 (doubly charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 600.29315 (doubly charged). Fig. 11 shows MS fragmentation spectra of peptides TPPSSGEPPK (SEQ ID NO: 12) and TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) (obtained with PRM). Diagnostic O-GlcNAc ions (indicated by solid arrows to the left) are predominantly lost upon fragmentation. Dotted arrows to the right indicate product ions with O-GlcNAc modification. Table 9 identifies product ions of TPPSSGEPPK*O-GlcNAc (SEQ ID NO: 16 or 17) peptide. The product ion y8 is listed in bold to reflect it containing O-GlcNAc. Product ion annotation based is based on O-GlcNAcylation at SI 85.
Table 9.
Figure imgf000045_0001
[00144] Even though high-quality fragmentation spectra were obtained, only limited amount of information was gathered for localization of the O-GlcNAc-site: one product ion with retention of the O-GlcNAc-moiety was observed, representing y8 fragmentation and thereby excluding O- GlcNAcylation at T181. Based on the available spectra, it was not possible to distinguish between (9-GlcN Acylation at S 184 or S 185.
[00145] A long tryptic peptide containing T123 and corresponding to sequence QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHV-T123-QAR (SEQ ID NO: 39), was detected with O-GlcNAcylation: the O-GlcN Acylated peptide eluted just before its unmodified form and signature peptides for O-GlcN Acylation (m/z 204, etc.) were observed. Product ions in accordance with the peptide sequence were detected, however all without an (9-GlcN Ac-moiety.
[00146] Fig. 12 shows extracted ion chromatograms of peptides QAAAQPHTEIPEGTTAE EAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAG HVTQAR*O-GlcNAc. The mass of the precursor ion non-modified peptide is at m/z 989.71897 (quadruply charged) and the mass of the precursor ion O-GlcNAcylated peptide is at m/z 1040.48881 (quadruply charged). Fig. 13 shows MS fragmentation spectra of peptides QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR (SEQ ID NO: 39) and QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR*O-GlcNAc. Diagnostic O- GlcNAc ions (indicated in solid arrow to the left) are predominantly lost upon fragmentation. Table
10 identifies product ions of QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVT QAR*O- GlcNAc peptide. Product ion annotation is based on O-GlcNAcylation at T123.
Table 10.
Figure imgf000046_0001
Figure imgf000047_0001
[00147] Consecutive trypsin - asp-N digestion of O-GlcNAc rec htau did not result in the discovery of smaller O-GlcNAc-peptides containing T123. Because of the long sequence of the ( -GlcN Ac-peptide and the availability of multiple potential O-GlcN Ac-sites, this peptide was not considered interesting in the search of O-GlcNAc-peptides in mouse BH. No O-GlcNAc-peptides containing S238 were detected in Example 2: this O-GlcNAc-site was also considered less interesting, as it is located in the C-terminal part of tau that is lost during truncation.
[00148] Based on the data generated in Example 2, the three following peptides were selected to for further analysis in mouse BH in Example 4 discussed below: SGYSSPGSPGTPG-(O- GlcNAc)S208-R (SEQ ID NO: 19), TPPSSGEPPK-(O-GlcNAc)S191-GDR(SEQ ID NO: 18) and TPPSSGEPPK*O-GlcNAc (at S184 or S185, most probably O-GlcNAcylated at S185)(SEQ ID NO: 16 or 17).
Example 3: Detection of D-GIcN Acylation sites in Recombinant Human Tau Using - elimination/Michael addition
[00149] Example 3 describes an exemplary method for selective clean-up and derivatization of O-GlcNAc rec htau in an effort to mitigate matrix effects from highly abundant unmodified peptides in tryptic digests, and to improve position identification by mitigating the easy loss of O- GlcNAc-moieties by in-source fragmentation. 0-elimination/Michael addition is used in Example 3 for the structural analysis of the O-glycosylated tau proteins. A process for 0- elimination/Michael addition of recombinant human tau protein is described below.
[00150] Unless indicated otherwise below, the materials used in Example 3 are the same as described above or Example 1.
Chemo- enzymatic derivatization of O-GlcNAcylated recombinant human tau
Chemo-enzymatic derivatization of O-GlcNAcylated recombinant human tau, trypsinization, enrichment, and release of tagged peptides by f-elimination - Michael addition reaction [00151] ( -GlcN Acylated tau is expressed in the same manner as described above in Example 2. The O-GlcN Acylated is then derivatized and purified. In particular, O-GlcNAc sites of O- GlcNAc rec htau were first chemo- enzymatically labelled with biotin, biotinylated O-GlcNAc rec htau was subsequently trypsinized, followed by immobilization/purification of biotinylated O- GlcNAc-peptides with streptavidin beads. Non-bound peptides were removed by washing the beads. Immobilized O-GlcNAc-peptides were released by transformation into free sulfited peptides via P-elimination - Michael addition reaction. A schematic representation of the derivatization and purification process 200 described below is provided in Figure 2.
[00152] 20 pL of 190 pg/mL O-GlcNAc rec htau was aliquoted to a low bind Eppendorf tube and precipitated with a chloroform/methanol/water washing procedure described in the ‘Click- iT™ O-GlcNAc Enzymatic Labeling System’ -protocol (MP 33368, Thermo Fisher Scientific). Briefly, 600 pL methanol, 150 pL chloroform and 400 pL water were added: the tube was vortex mixed for 5 s after each addition. The mixture was centrifuged for 5 minutes at 18,000/g (Heraeus Megafuge 8R centrifuge, Thermo Fisher Scientific). The upper aqueous phase was then carefully removed as much as possible and discarded while leaving the interface layer containing the protein precipitate intact. 450 pL of methanol was added, the tube was vortex mixed and then centrifuged for 5 minutes at 18,000/g to pellet the protein. The supernatant was removed and discarded, and the procedure was repeated once more. The pellet was allowed to air-dry. The protein was resuspended in 40 pL of 1% SDS in 20 mM HEPES pH 7.9, heated for 8 min at 90°C while shaking (ThermoMixer, Thermo Fisher Scientific) to dissolve the pellet, and immediately cooled on melting ice.
[00153] In step 204, enzymatic labeling of O-GlcNAc rec htau with UDP-N- azidoacetylgalactosamine (UDP-GalNAz) and Gal-Tl (Y289L) was carried out by using the ‘Click-iT™ O-GlcNAc Enzymatic Labeling System’-kit (MP 33368, Thermo Fisher Scientific). After GalNAz-addition by overnight incubation at 4°C, modified O-GlcNAc rec htau was precipitated with the above chloroform/methanol/water clean-up. The protein pellet was dissolved in 50 pL 50 mM Tris pH 8.0 + 1% octyl P-D-glucopyranoside by heating for 8 min at 90°C while shaking, and immediately cooled on melting ice.
[00154] In step 206, iotinylation was performed via Click-iT™ azide/alkyne reaction by following the ‘Click- it Protein Analysis Detection Kits ’-protocol (MP 33372, Thermo Fisher Scientific). After incubating the reaction mixture for 30 min at room temperature, the GalNAzylated and biotinylated O-GlcNAc rec htau was precipitated with the above chloroform/methanol/water clean-up. The pellet was dissolved by heating for 8 min at 90°C while shaking in 150 pL 50 mM ammonium bicarbonate. After cooling on melting ice, 10 pL of acetonitrile was added followed by briefly vortex mixing.
[00155] For digestion in step 208, 25 pL of 0.05 mg trypsin/mL in 50 mM acetic acid was added and the sample was subsequently incubated at 37 °C for 20 h while shaking at 1,000 rpm (ThermoMixer, Thermo Fisher Scientific).
[00156] For selective capture of biotinylated O-GlcNAc tryptic tau peptides in step 210, the digest was incubated with Dynabeads™ M-280 Streptavidin (Thermo Fisher Scientific): 158 pL of 10 mg beads/mL in PBS pH 7.4 with 0.1% BSA and 0.02% sodium azide was aliquoted in a LoBind Eppendorf tube, placed on a DynaMag™-2 Magnet, solvent was removed and the beads were washed in with 1 mL of DPBS + 0.1% octyl P-D-glucopyranoside by vortex mixing for 5 s. The tube was spun down and placed in a DynaMag™-2 Magnet and the supernatant was discarded. The rec htau digest containing biotinylated GalNAz-O-GclNAc-peptides was evaporated with nitrogen gas at 45°C (not to completely dry), dissolved in 200 pL DPBS and transferred to the streptavidin beads. The mixture was incubated at room temperature for 30 min using gentle rotation. The tube was placed on a magnet and the depleted fraction was transferred and stored for analysis. The coated beads were washed four times with 400 pL DPBS + 0.1% BSA and once more with 400 pL DPBS.
[00157] Finally, in step 212, P-eliminati on -Michael addition reaction was performed to release derivatized O-GlcNAc tryptic tau peptides as sulfited peptides: DPBS was discarded, 100 pL of 0.1 N NaOH and 0.6 M Na2SCh were added, followed by incubation of the beads at room temperature for 24h. The reaction was stopped by adding 50 pL of 0.3 M acetic acid.
LC-MS nLC-HRMS analysis of purified sulfited peptides from O-GlcNAcylated recombinant human tau
[00158] Method settings for nLC-HRMS analysis of (9-GlcN Acylated recombinant human tau digests as described above in Examples 1 and 2, were used for the analysis of the sulfited peptides of Example 3, with some modifications: 20 pL of extract was injected and trapping was prolonged to 5 min to wash away salts present in the extract before coupling the trap column in line with the nLC-column. The first part of the nLC-gradient (from 2% B to 28% B) was thereby shortened from 24 mm to 21 mm. For peptides SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24), TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23) and TPPSSGEPPK*SO3 representing tau sulfite modification at either SI 84 or SI 85 (SEQ ID NO: 21 or 22) additional PRM experiments were set up. Maximum injection times were changed to 500 ms, 500 ms and 3000 ms, respectively. The doubly charged peptides with mlz 729.30171, mlz 738.32519 and mlz 530.73441, respectively, were added to an inclusion list for fragmentation.
Results and Discussion
Derivatization strategy to enrich O-GlcNAc modified peptides of O-GlcNAcylated recombinant human tau
[00159] Initial P-elimination/Michael addition derivatization in Example 3 with standard dilutions of SPVV-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) and SPWSGDTSPR (SEQ ID NO: 15) showed almost complete conversion of SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) into SPW-(SO3)S-GDTSPR (SEQ ID NO: 25), and fragmentation on HRMS provided diagnostic product ions for the site of modification. In the extract of SPWSGDTSPR (SEQ ID NO: 15) after derivatization however, artifact formation was observed: approximately 3% of SPWSGDTSPR (SEQ ID NO: 15) was converted to SPW-(SO3)S-GDTSPR (SEQ ID NO: 25), even though the standard dilutions were subjected to mild derivatization conditions. As ( -GlcN Acylated tau is present in low stoichiometry in the brain, the relative amount of artifacts formed by sulfite addition to non-modified S/T-sites in tau would be significant and lead to false positive identifications. Next to that, other tau PTMs like phosphorylation would be derivatized as well, emphasizing the need for selective clean-up of (9-GlcN Acylated peptides prior to P-elimination/sulfite addition.
[00160] (9-GlcNAc rec htau was subjected to the above-described purification strategy (and also shown in Fig. 14) and LC-HRMS was used to monitor reaction products of tau O- GlcNAcylated at S400 after several of the reaction steps. For the generation of the data of Figs. 15-20, the same chromatographic conditions were applied as used during UHPLC-MSMS analysis of brain homogenate extracts for the detection of tau S400 O-GlcNAcylation in Example 1, except that mobile phase solvents consisted of A: water + 0.1% FA and B: acetonitrile + 0.1% FA. The UHPLC-system was hyphenated with a TripleTOF (6600, Sciex). The peptides were ionized with electrospray ionisation (ESI) in positive ion mode. The ionspray voltage was set to 4500 V, temperature to 400°C, declustering potential to 80 V. Ion source gas 1, gas 2 and curtain gas were set to 50, 40 and 30, respectively. CAD gas was set to 6. Data were acquired in TOF MS-mode with m/z-range set to measure from mlz 100 to mlz 1500. Table 11 below shows the exact mass and charge states of each of the intermediates of the purification process of the derivatization and purification process of Fig. 14.
Table 11.
Figure imgf000051_0001
[00161] After trypsinization of GalNAzylated and biotinylated O-GlcNAc rec htau for instance, extracted ion chromatograms of SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S- GDTSPR (SEQ ID NO: 20), SPW-(O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc- GalNAz-biotin)S-GDTSPR confirmed that almost all SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20) was converted. Fig. 15 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S-GDTSPR (SEQ ID NO: 20), SPW-(O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR (20 mDa mass extraction window) in the extract of GalNAzylated, biotinylated and trypsinized O- GlcNAc rec htau. SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR was also observed in Fig. 15, which reassured that biotinylation was successful. However, detection of SPW-(O-GlcNAc- GalNAz)S-GDTSPR (which partially fragmented in the MS source to render SPW-(O- GlcNAc)S-GDTSPR (SEQ ID NO: 20)) revealed that not all substrate for biotinylation had been converted. Also the depleted fraction after incubation with streptavidin beads was examined this way: except for SPW-(O-GlcNAc-GalNAz-biotin)S-GDTSPR, all the above peptides were detected, confirming capture of the biotinylated peptides. Fig. 16 shows a zoom-in of extracted ion chromatograms of tryptic peptides SPWSGDTSPR (SEQ ID NO: 15), SPW-(O-GlcNAc)S- GDTSPR (SEQ ID NO: 20), SPW-(O-GlcNAc-GalNAz)S-GDTSPR and SPW-(O-GlcNAc- GalNAz-biotin)S-GDTSPR (20 mDa mass extraction window) in the depleted fraction after capture of GalNAzylated, biotinylated and trypsinized O-GlcNAc rec htau peptides with streptavidin beads.
[00162] After the final derivatization step, namely P-elimination - Michael addition reaction to release the biotinylated peptides as sulfited peptides, the extracts were examined for potential artifacts (e.g., peptides with 2 sulfite moieties, etc.) with LC-HRMS: no artifacts were detected. Furthermore, almost no non-modified tryptic peptides were detected, indicating the high selectivity of the applied purification protocol. The extract containing the enriched sulfited peptides was analyzed with nLC-HRMS.
[00163] Fig. 17 shows an extracted ion chromatogram of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25)(6 ppm mass extraction window). The mass of SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) is at m/z 583.26133 (doubly charged). Fig. 18 shows MS fragmentation spectrum of SPW- (SO3)S-GDTSPR (SEQ ID NO: 25). Dotted arrows indicate product ions with sulfite modification. Table 12 identifies product ions of the SPW-(SO3)S-GDTSPR (SEQ ID NO: 25) peptide. The product ions b5, b7, y7, y8 and y9 are listed in bold to reflect those being product ions that contain sulfite moiety.
Table 12.
Figure imgf000052_0001
[00164] MS fragmentation of a sulfited SPWSGDTSPR (SEQ ID NO: 25) peptide confirmed O-GlcNAcylation at S400 in rec ( -GlcNAc htau, as both a b5 and y7 ion containing a sulfite moiety were detected. No diagnostic product ions were detected for the O-GlcNAcylation site when fragmenting a standard dilution of SPW-(O-GlcNAc)S-GDTSPR. The peak intensities of the detected sulfited peptides were less intense than the previously observed N-terminal and middomain O-GlcNAc peptides. Because of the low abundant peaks, it was decided to acquire data in PRM-mode to increase sensitivity.
[00165] Fig. 19 shows MS fragmentation spectrum of SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24). The dotted arrows indicate product ions with sulfite modification. The mass of SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24) is at m/z 729.30171 (doubly charged). Table 13 identifies product ions of the SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24) peptide. The product ions y2, y3, y4, y7-H2O, y7, ylO-H20 and ylO are listed in bold to reflect those being product ions that contain sulfite moiety.
Table 13.
Figure imgf000053_0001
[00166] As demonstrated by the data provide above, for peptide SGYSSPGSPGTPG-(SO3)S-R (SEQ ID NO: 24), several diagnostic product ions containing a sulfite moiety were observed, namely y2, y3 and y4, confirming O-GlcNAcylation at S208.
[00167] Fig. 20 shows MS fragmentation spectrum of TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23). The dotted arrows indicate product ions with sulfite modification. The mass for TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23) is at m/z 738.32519 (doubly charged). Table 14 identifies product ions of the TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23) peptide. The product ions y4, yl2, yl3, y6, yl4, y7 and yl2 are listed in bold to reflect those being product ions that contain sulfite moiety.
Table 14.
Figure imgf000054_0001
[00168] As shown in the data above, for peptide TPPSSGEPPK-(SO3)S-GDR (SEQ ID NO: 23) several product ions containing a sulfite moiety were detected, y4 being diagnostic for O- GlcN Acylation at S 191. [00169] For peptide TPPSSGEPPK*SO3 (SEQ ID NO: 21 or 22) however, even with a max injection time of 3000 ms, still no diagnostic product ions were detected. Moreover, the spectra were contaminated with product ions originating from a co-eluting isobaric interference (data not shown). No additional information was thus gathered regarding (9-GlcN Acylation at either SI 84 or S185. [00170] In summary, nLC-HRMS analysis of the enriched sulfited peptides rendered complementary information and confirmation of the identified O-GlcN Ac-sites in Example 2. However, due to the extensive sample preparation procedure and the observed low intensity of analyte peaks, Example 4 (described below) analyzed mouse BH digests without 0- elimination/Michael addition derivatization.
Example 4: IP-LC-MSMS of tau U-GIcN Acylation Site in Mouse Brain Homogenates [00171] In Example 4, the mouse homogenates obtained in Example 1 are enzymatically digested according to Example 2 and further analyzed using UHPLC-MSMS and nLC-MSMS according to the methods below. Unless indicated otherwise below, the materials used in Example 4 are the same as described above or Example 1. The same mouse homogenates obtained in Example 1 are further analyzed in Example 4 according to the methods described below.
LC-MS UHPLC-MSMS analysis of brain homogenate extracts for the detection of new O-
GlcNAcylation sites of tau
[00172] Method settings for UHPLC-MSMS analysis of brain homogenate extracts for the detection of tau S400 O-GlcNAcylation as described above, were used for the identification of new tau ( -GlcN Acylation sites in mouse brain homogenate, with some modifications: ionspray voltage was set at 4500 V, temperature to 400°C and declustering potential to 60 V. The selected MRM-transitions are provided below. Table 15 lists the MRM-transitions used in UHPLC-MSMS experiments for the detection of new O-GlcNAcylation sites in mouse brain homogenate samples.
Table 15.
Figure imgf000055_0001
nLC-MSMS analysis of mouse brain homogenates
[00173] Before analysis, 10 pL 1% TFA in water was added to 80 pL of mouse brain homogenate sample extracts. Chromatographic analysis was performed on a Thermo Fisher Scientific Ultimate 3000 RSLC nano LC System with Pro Flow technology (Thermo Fisher Scientific). 50 pL of diluted BH digest was injected and loaded on an Acclaim™ PepMap™ 100 C18 trap column, 5 pm, 0.3 mm x 5 mm (Thermo Fisher Scientific): 100:0.5:0.1 water:acetonitrile:FA was pumped isocratically at 10 pL/min with a pLC loading pump, diverting the eluate of the trap column to waste. A 10-port switching valve was used to divert the eluate of the trap column to waste. After 6 min of sample loading, the valve was switched to elute the trapped analytes in backflush mode onto a nanoEase MZ HSS T3 Column, 100 A, 1.8 pm, 75 pm x 150 mm (Waters). The analytes were chromatographed with a linear gradient at 300 nL/min provided by a nLC binary pump, with mobile phases A: water + 0.1% FA, and B: 20:80:0.1 water:acetonitrile:FA. Details of the chromatographic conditions are provided in Table 16.
[00174] Table 16 provides LC-gradient and valve switching timing of the nLC-MSMS method used for analysis of O-GlcNAcylated tryptic tau peptides in extracts of mouse brain homogenates. Mobile phase solvents A and B are pumped by the nLC binary pump, mobile phase solvent C is provided by the pLC loading pump. Valve position ‘O’: pLC flow passes via sample loop through the trap column and finally elutes to waste (load position). Valve position ‘1 ’: nLC flow passes through trap column and analytical column to finally enter the MS system. The column ‘Parameter’ indicates valve position (0 or 1), and percentage (%) B and C solvent, respectively.
Table 16.
Figure imgf000056_0001
[00175] Both columns and the 10-port switching valve were heated to 40° C in a column thermostat. The eluant of the nLC column was transferred via an Optiflow turbo V ion source (Sciex) to a 6500+ mass spectrometer (Sciex). The peptides were ionized with electrospray ionisation (ESI) in positive ion mode. The ionspray voltage was set to 3000 V, temperature to 150°C, declustering potential to 60 V and entrance potential to 10 V. Ion source gas 1, gas 2 and curtain gas were set to 10, 0 and 25, respectively. CAD gas was set to 6. The selected MSMS transitions used for multiple reaction monitoring of both non-modified and O-GlcNAcylated peptides are provided below. Table 17 lists MRM-transitions used in nLC-MSMS experiments for the detection of new O-GlcNAcylation sites in mouse brain homogenate samples. Table 17.
Figure imgf000057_0001
[00176] Thermo Scientific Dionex Chromeleon Chromatography Data System software (version 7.2.7) was used for operating the nLC system. Analyst 1.7 (Sciex) was used for operating the MS system. MS acquisition was initiated by the nLC instrument via a start-stop signal. Data processing was performed with Sciex OS 1.6 (Sciex). Results and Discussion
[00177] The UHPLC-MSMS method used for the detection of S400 (9-GlcN Acylation in mouse BH samples was adjusted to monitor the three N-terminal and mid-domain tau O-GlcNAcylation sites in mouse BH digests: 3 MRM-transitions were selected per O-GlcNAc-peptide, based on previously obtained nLC-HRMS fragmentation spectra of an O-GlcNAc rec htau digest in Example 2 (see Table 15). Product ions diagnostic for the location of the O-GlcNAc-moiety were included. As no reference compounds were available, collision energy was set at an arbitrary value of 30.
[00178] Immunoprecipitated and digested tau from non-treated and Thiamet-G treated mouse BH were analyzed, an O-GlcNAc rec htau digest was injected as reference material. All three tau N-terminal and mid-domain O-GlcNAc-peptides identified in Example 2 were detected in the O- GlcNAc rec htau digest.
[00179] Fig. 21A shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either S 184 or S 185, originating from IP-ed and digested tau from a brain homogenate of Thiamet-G treated mice. Fig. 21B shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85, originating from IP-ed and digested tau from a brain homogenate of non-treated mice. Fig. 21 C shows UHPLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc, representing tau O-GlcNAcylation at either SI 84 or SI 85, originating from a digest of O-GlcNAc recombinant human tau.
[00180] In the UHPLC-MSMS chromatograms of mouse BH samples, only TPPSSGEPPK*O- GlcNAc was detected, although at low intensity, in its 3 selected transitions in the Thiamet-G treated mouse BH digest, thereby confirming the presence of tau ( -GlcN Acylation at either SI 84 or SI 85 in mouse brain when treated with Thiamet-G. The peptide was not observed in the nontreated mouse BH sample.
[00181] To augment sensitivity, the nLC-MSMS method described above was developed. MRM transitions to monitor non-modified tryptic tau peptides were added to improve the quality of analysis. For Example 4, only a limited amount of sample extracts originating from Studyl and Study2 remained for analysis: immunoprecipitated and digested tau from Thiamet-G treated mouse BH, a method blank (IP-ed and digested buffer H) and an extract obtained from tauKO mouse BH were analysed. [00182] Fig. 22A shows nLC-MSMS chromatograms of SPW-((0-GlcNAc)S400)-GDTSPR and SPWSGDTSPR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G. Fig. 22B shows nLC-MSMS chromatograms of SGYSSPGSPGTPG-(O-GlcNAc)S208-R and SGYSSPGSPGTPGSR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G. Fig. 22C shows nLC-MSMS chromatograms of TPPSSGEPPK-(O-GlcNAc)S191-GDR and TPPSSGEPPKSGDR originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G. Fig. 22D shows nLC-MSMS chromatograms of TPPSSGEPPK*O-GlcNAc (most probably O-GlcNAcylated at SI 85) and TPPSSGEPPK originating from IP-ed and digested tau from a brain homogenate of mice treated with a high dose of Thiamet-G.
[00183] Table 18 identifies peak areas of monitored surrogate tryptic peptides of tau, with and without O-GlcN Acylation, detected in brain homogenates of P301S transgenic mice from 2 independent Thiamet G treatment studies (Study 1 and Study 2), a method blank (IP-ed and digested buffer H) and in a brain homogenate of tau KO mice based on data obtained using IP-nLC-MSMS.
The data in Table 18 presented in italics indicate peak areas detected due to carryover or an interference.
Table is.
Figure imgf000059_0001
[00184] In contrast with UHPLC-MSMS analysis, the three targeted O-GlcNAc peptides were detected in all O-GlcNAc-specific MRM transitions in BH extracts of mice treated with a high dose of Thiamet G, thereby confirming the presence of tau O-GlcNAcylation at SGYSSPGSPGTPG-(6»-GlcNAc)S208-R, TPPSSGEPPK-(O-GlcNAc)S 191 -GDR and
TPPSSGEPPK*O-GlcNAc (most probably O-GlcN Acylated at S185) (see Table 18).
[00185] The peak areas obtained for the high dose samples from Study 1 and Study2 were similar. For the low and high Thiamet G dose BH samples from Study2, non-modified peptide peak areas were comparable, while lower peak areas were obtained for the ( -GlcN Ac peptides in the low dose samples. These data seem to suggest an effect of Thiamet G on O-GlcNAcylation of the examined N-terminal and mid domain O-GlcNAc sites in tau.
[00186] Some minor peaks were detected in the MRM traces of non-modified peptides in the method blank and tau KO sample extracts: these can be explained by carryover from the previously injected POC sample extracts containing relatively high concentrations of non-modified peptides (see Table 18). Next to that, in the tau KO sample extract minor peaks were detected in some of the MRM-transitions representing O-GlcNAc peptides. Nonetheless, for each of the three newly detected N-terminal and mid domain O-GlcNAc-peptides no peak was detected in at least one of the MRM-transitions, thereby confirming selectivity. Finally, as observed previously during nLC analysis of TPPSSGEPPI<-(G-GlcNAc)S l 91 -GDR and TPPSSGEPPK*O-GlcNAc and their nonmodified counterparts, broad chromatographic peaks were observed.
[00187] To conclude, IP-nLC-MSMS analysis of BH extracts of transgenic P301S mice treated with Thiamet G enabled identification of low levels of O-GlcNAcylation in the N-terminal and mid domain of tau, namely at positions S208, S191 and S184 or S185.
[00188] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed since these embodiments are intended as illustrations of several aspects of this invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety. Sequences
SEQ ID NO : 1 - human tau protein 2N4R
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAWR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQI INK KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGS IDMV DSPQLATLAD EVSASLAKQG L
SEQ ID NO : 2 - PT9 HCDR1
GYTFTNYW
SEQ ID NO : 3 - PT9 HCDR2
IDPSDSYT
SEQ ID NO : 4 - PT9 HCDR3
GNLRGY
SEQ ID NO : 5 - PT9 LCDR1
QSIVDSNGNTY
SEQ ID NO : 6 - PT9 LCDR2
KVF
SEQ ID NO : 7 - PT9 LCDR3
FQGSHVPYT
SEQ ID NO : 8 - VH of PT9 antibody
QVQLQQPGAE FVKPGASVKL SCKASGYTFT NYWMQWVKQR PGQGLEWIGE IDPSDSYTNY NQNFKGKATL TVDTSSSTAY MQLSSLTSED SAVYYCGNLR GYWGQGTTLT VSS
SEQ ID NO : 9 - VL of PT9 antibody
DVLMTQTPLS LPVSLGDQAS ISCRSSQS IV DSNGNTYLEW YQQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP YTFGGGTKLE IK SEQ ID NO : 10 - heavy chain of PT9 antibody
QVQLQQPGAE FVKPGASVKL SCKASGYTFT NYWMQWVKQR PGQGLEWIGE IDPSDSYTNY NQNFKGKATL TVDTSSSTAY MQLSSLTSED SAVYYCGNLR GYWGQGTTLT VSSAKTTAPS VYPLAPVCGD TTGSSVTLGC LVKGYFPEPV TLTWNSGSLS SGVHTFPAVL QSDLYTLS SS VTVTSSTWPS QS ITCNVAHP ASSTKVDKKI EPRGPT IKPC PPCKCPAPNL LGGPSVFI FP PKIKDVLMI S LSPIVTCVW DVSEDDPDVQ I SWFVNNVEV HTAQTQTHRE DYNSTLRWS ALPIQHQDWM SGKEFKCKVN NKDLPAPIER TI SKPKGSVR APQVYVLPPP EEEMTKKQVT LTCMVTDFMP EDIYVEWTNN GKTELNYKNT EPVLDSDGSY FMYSKLRVEK KNWVERNSYS CSWHEGLHN HHTTKSFSRT PGK
SEQ ID NO : 11 - light chain of P' 9 antibody
DVLMTQTPLS LPVSLGDQAS I SCRSSQS IV DSNGNTYLEW YQQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP YTFGGGTKLE IKRADAAPTV S I FPPSSEQL TSGGASWCF LNNFYPKDIN VKWKI DGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC
SEQ ID NO : 12 - Amino acids 181-190 of human tau protein 2N4R
TPPSSGEPPK
SEQ ID NO : 13 - Amino acids 181-194 of human tau protein 2N4R
TPPSSGEPPKSGDR
SEQ ID NO : 14 - Amino acids 195-209 of human tau protein 2N4R SGYSS PGSPGTPGSR
SEQ ID NO : 15 - Amino acids 396-406 of human tau protein 2N4R
SPWSGDTSPR
SEQ ID NO : 16 - Amino acids 181-190 of human tau protein 2N4R O- GlcNAcylated at S184
TPP- ( O-GlcNAc ) S-SGEPPK
SEQ ID NO : 17 - Amino acids 181-190 of human tau protein 2N4R O- GlcNAcylated at S185
TPPS- ( O-GlcNAc ) S-GEPPK SEQ ID NO: 18 - Amino acids 181-194 of human tau protein 2N4R O- GlcNAcylated at SI 91
TPPSSGEPPK- (O-GlcNAc) S-GDR
SEQ ID NO: 19 - Amino acids 195-209 of human tau protein 2N4R O-
GlcNAcylated at S208
SGYSSPGSPGTPG- (O-GlcNAc) S-R
SEQ ID NO: 20 - Amino acids 396-406 of human tau protein 2N4R O- GlcNAcylated at S400
SPW- (O-GlcNAc) S-GDTSPR
SEQ ID NO: 21 - Amino acids 181-190 of human tau protein 2N4R sulfite modified at S184
TPP- (S03) S-SGEPPK
SEQ ID NO: 22 - Amino acids 181-190 of human tau protein 2N4R sulfite modified at S185
TPPS- (S03) S-GEPPK
SEQ ID NO: 23 - Amino acids 181-194 of human tau protein 2N4R sulfite modified at S191
TPPSSGEPPK- (S03) S-GDR
SEQ ID NO: 24 - Amino acids 195-209 of human tau protein 2N4R sulfite modified at S208
SGYSSPGSPGTPG- (S03) S-R
SEQ ID NO: 25 - Amino acids 396-406 of human tau protein 2N4R sulfite modified at S400
SPW- (S03) S-GDTSPR
SEQ ID NO: 26 - Synthetic peptide
H-SPW- (O-GlcNAc) S-GDTSPR-OH
SEQ ID NO: 27 - Synthetic peptide
H-SPWSGDTSPR-OH SEQ ID NO: 28 — Synthetic peptide
H-TPSLPTPPTR-OH
SEQ ID NO: 29 - Phosphorylated (pT) synthetic peptide
H-TPSLP (pT) PPTR-OH
SEQ ID NO: 30 - Phosphorylated (pT) synthetic peptide
H-SR(pT) PSLP (pT) PPTREPK-OH
SEQ ID NO: 31 - Amino acids 212-221 of human tau protein (*: Arginine labelled with 13C and 15N)
H-TPSLPTPPTR*-OH
SEQ ID NO: 32 -- Amino acids 212-221 of human tau protein phosphorylated at residue 217 (*: Arginine labelled with 13C and
Figure imgf000064_0001
SEQ ID NO: 33 - Amino acids 396-406 of human tau protein 2N4R SPWSGDTSPR
SEQ ID NO: 34 - Amino acids 212-221 of human tau protein 2N4R phosphorylated at residue 217
TPSLP (pT) PPTR
SEQ ID NO: 35 -- Amino acids 21-241 of human tau protein 2N4R phosphorylated at residues 212 and 217
SR(pT) PSLP (pT) PPTREPK
SEQ ID NO: 36 -- Amino acids 212-221 of human tau protein 2N4R
TPSLPTPPTR
SEQ ID NO: 37 - human OGT Protein
MASSVGNVAD STGLAELAHR EYQAGDFEAA ERHCMQLWRQ EPDNTGVLLL LSSIHFQCRR LDRSAHFSTL AIKQNPLLAE AYSNLGNVYK ERGQLQEAIE HYRHALRLKP DFIDGYINLA AALVAAGDME GAVQAYVSAL QYNPDLYCVR SDLGNLLKAL GRLEEAKACY LKAIETQPNF AVAWSNLGCV FNAQGEIWLA IHHFEKAVTL DPNFLDAYIN LGNVLKEARI FDRAVAAYLR ALSLSPNHAV VHGNLACVYY EQGLIDLAID TYRRAIELQP HFPDAYCNLA NALKEKGSVA EAEDCYNTAL RLCPTHADSL NNLANIKREQ GNIEEAVRLY RKALEVFPEF AAAHSNLASV LQQQGKLQEA LMHYKEAIRI SPTFADAYSN MGNTLKEMQD VQGALQCYTR AIQINPAFAD AHSNLAS IHK DSGNI PEAIA SYRTALKLKP DFPDAYCNLA HCLQIVCDWT DYDERMKKLV S IVADQLEKN RLPSVHPHHS MLYPLSHGFR KAIAERHGNL CLDKINVLHK PPYEHPKDLK LSDGRLRVGY VSSDFGNHPT SHLMQS I PGM HNPDKFEVFC YALSPDDGTN FRVKVMAEAN HFIDLSQI PC NGKAADRIHQ DGIHILVNMN GYTKGARNEL FALRPAPIQA MWLGYPGTSG ALFMDYI ITD QETSPAEVAE QYSEKLAYMP HTFFIGDHAN MFPHLKKKAV IDFKSNGHIY DNRIVLNGID LKAFLDSLPD VKIVKMKCPD GGDNADSSNT ALNMPVI PMN TIAEAVIEMI NRGQIQIT IN GFS I SNGLAT TQINNKAATG EEVPRT I IVT TRSQYGLPED AIVYCNFNQL YKIDPSTLQM WANILKRVPN SVLWLLRFPA VGEPNIQQYA QNMGLPQNRI I FSPVAPKEE HVRRGQLADV CLDTPLCNGH TTGMDVLWAG TPMVTMPGET LASRVAASQL TCLGCLELIA KNRQEYEDIA VKLGTDLEYL KKVRGKVWKQ RI SS PLFNTK QYTMELERLY LQMWEHYAAG NKPDHMIKPV EVTESA
SEQ ID NO : 38 - Amino acids 396-406 of human tau protein 2N4R
HLSNVSSTGS I
SEQ ID NO : 39 - Amino acids 98-126 of human tau protein 2N4R
QAAAQPHTEI PEGTTAEEAGIGDTPSLEDEAAGHVTQAR

Claims

CLAIMS What is claimed is:
1. A method for detecting O-GlcNAc hydrolase (OGA) inhibition in a brain of a subject, the method comprising: a) detecting O-GlcNAcylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein to determine an amount of O-GlcN Acylated tau peptides in a biologic sample obtained from the subject, wherein OGA inhibition is determined to be present in the brain of the subject when the amount of O-GlcN Acylated tau peptides is above a predetermined threshold value.
2. The method of claim 1, wherein the biologic sample is cerebrospinal fluid (CSF).
3. The method of claim 1, wherein an OGA inhibitor was administered to the subject.
4. The method of claim 1 , wherein step a) comprises detecting O-GlcN Acylation in (i) residue
184 or 185, (ii) residue 191 , (iii) residue 208, and (iv) residue 400 of the tau protein to determine an amount of (9-GlcN Acylated tau peptides in a biologic sample obtained from the subject.
5. The method of claim 1, wherein step a) comprises detecting O-GlcNAcylation in at least one of residue 184, 185, 191, and 208 of the tau protein to determine an amount of O- GlcN Acylated tau peptides in a biologic sample obtained from the subject.
6. The method of claim 5, wherein step a) comprises detecting (9-GlcN Acylation in (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 of the tau protein to determine an amount of O- GlcN Acylated tau peptides in a biologic sample obtained from the subject.
7. The method of any one of claims 1 -6, wherein the O-GlcN Acylation is detected by liquid chromatography mass spectrometry (LC-MS) or an immunoassay.
64
RECTIFIED SHEET (RULE 91 ) ISA/EP
8. The method of claim 7, wherein the LC-MS is nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS), liquid chromatography - tandem mass spectrometry (LC-MS/MS), nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS), or ultra-high performance liquid chromatography- MS/MS (UHPLC-MSMS).
9. The method of claim 7, wherein the immunoassay is a single molecule array (SIMOA) assay.
10. The method of any one of claims 1-6, wherein the tau protein in the biologic sample is concentrated by immunoprecipitation before the detecting of ( -GlcN Acylation.
11. The method of claim 10, wherein the G-GIcN Acylation is detected by liquid chromatography mass spectrometry (LC-MS) or immunoassay.
12. The method of claim 11, wherein the LC-MS is nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS), liquid chromatography - tandem mass spectrometry (LC-MS/MS), nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS) or ultra-high performance liquid chromatography- MS/MS (UHPLC-MSMS).
13. The method of claim 11, wherein the immunoassay is a single molecule array (SIMOA) assay.
14. An assay method of detecting O-GlcNAc-tau peptides, said method comprising: obtaining a biologic sample from a human subject; contacting the biologic sample with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support to isolate tau protein from the biologic sample; digesting the isolated tau protein with at least one enzyme; and
65
RECTIFIED SHEET (RULE 91 ) ISA/EP detecting O-GlcN Acylation in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry to determine an amount of O- GlcNAcylated tau peptides in the biologic sample.
15. The assay method of claim 14, wherein the biologic sample is cerebrospinal fluid (CSF).
16. The assay method of claim 14, wherein the solid support is magnetic beads.
17. The assay method of claim 14, wherein the immunoprecipitation antibody binds to an epitope between ammo acids 163 to 174 of human tau protein or an epitope between ammo acids
219 to 226 of the human tau protein.
18. The assay method of claim 14, wherein the immunoprecipitation antibody comprises an immunoglobulin heavy chain HCDR1 , HCDR2 and HCDR3 comprising the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively and an immunoglobulin light chain LCDR1, LCDR2 and LCDR3 comprising the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively.
19. The assay method of claim 18, wherein the immunoglobulin heavy chain comprising the polypeptide sequence of SEQ ID NO: 8 and the immunoglobulin light chain comprises the polypeptide sequence of SEQ ID NO: 9.
20. The assay method of claim 19, wherein the immunoprecipitation antibody is PT9.
21. The assay method of claim 14, wherein (9-GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 are detected.
22. The assay method of claim 21, wherein O-GlcNAcylation at (i) residue 184 or 185, (ii) residue 191 , (iii) residue 208, and (iv) residue 400 are detected.
66
RECTIFIED SHEET (RULE 91 ) ISA/EP
23. The assay method of claim 14, wherein the liquid chromatography mass spectrometry is nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS), liquid chromatography - tandem mass spectrometry (LC-MS/MS), or nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS).
24. The assay method of claim 14, wherein the isolated tau protein is digested with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested peptides.
25. The assay method of claim 24, wherein the isolated tau protein is divided into at least five aliquots and digested with the at least one enzyme, wherein the aliquots comprise:
(1) an aliquot digested with trypsin;
(2) an aliquot digested with asp-N;
(3) an aliquot digested with glu-C;
(4) an aliquot digested with trypsin followed by asp-N; and
(5) an aliquot digested with trypsin followed by glu-C.
26. The assay method of claim 25, wherein O-GIcNAcylation at (i) residue 184 or 185, (ii) residue 191, and (hi) residue 208 are detected.
27. The assay method of claim 26, wherein (9-GlcNAcylation at (i) residue 184 or 185, (ii) residue 191, (iii) residue 208, and (iv) residue 400 are detected.
28. The assay method of claim 26, wherein (9-GlcNAcylation at (i) residue 184 or 185 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 10 or 11.
29. The assay method of claim 26, wherein O-GIcN Acylation at (ii) residue 191 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 12.
30. The assay method of claim 26, wherein (9-GlcNAcylation at (iii) residue 208 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 13.
67
RECTIFIED SHEET (RULE 91 ) ISA/EP
31. The assay method of claim 27, wherein O-GlcN Acylation at (iv) residue 400 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 14.
32. The assay method of claim 28, wherein (9-GlcN Acylation at (i) residue 184 or 185 is detected by detecting ion transition from 600.3 m/z to 1001.5 m/z, 600.3 m/z to 996.5 m/z, or from 600.3 m/z to 798.4 m/z.
33. The assay method of claim 29, wherein O-GIcNAcylation at (ii) residue 191 is detected by detecting ion transition from 538.9 m/z to 959.5 m/z, or 538.9 m/z to 756.4 m/z.
34. The assay method of claim 30, wherein O-GlcNAcylation at (ii) residue 208 is detected by detecting ion transition from 798.9 m/z to 619.3 m/z, 798.9 m/z to 874.4 m/z, or from 798.9 m/z to 1115.5 m/z.
35. The assay method of claim 31, wherein (9-GlcNAcylation at (iv) residue 400 is detected by detecting ion transition from 652.8 m/z to 1101.4 m/z.
36. An assay method of detecting O-GlcNAc-tau peptides, said method comprising: obtaining a biologic sample from a human subject; contacting the biologic sample with an immunoprecipitation antibody directed against tau protein to bind the immunoprecipitation antibody to tau protein in the biologic sample to form antibody-peptide complexes binding to a solid support to isolate tau protein from the biologic sample; labelling ( -GlcNAc sites of the isolated tau protein with biotin to produce biotinylated O-GlcNAc peptides; digesting the biotinylated O-GlcNAc peptides with at least one enzyme selected from the group consisting of: trypsin, asp-N, and glu-C, to produce digested biotinylated peptides; immobilizing the digested biotinylated peptides to a solid support; reacting the digested biotinylated peptides with Na2SCh in a P-elimination - Michael addition reaction to release sulfited peptides from the solid support;
68
RECTIFIED SHEET (RULE 91 ) ISA/EP detecting sulfite modification in at least one of residue 184, 185, 191, 208, and 400 of the tau protein by liquid chromatography mass spectrometry to determine an amount of O- GlcNAcylated tau peptides in the biologic sample.
37. The assay method of claim 36, wherein the biologic sample is cerebrospinal fluid (CSF).
38. The assay method of claim 36, wherein the solid support is streptavidin beads.
39. The assay method of claim 36, wherein the immunoprecipitation antibody binds to an epitope between ammo acids 163 to 174 of human tau protein or an epitope between ammo acids 219 to 226 of the human tau protein.
40. The assay method of claim 36, wherein the immunoprecipitation antibody comprises an immunoglobulin heavy chain HCDR1 , HCDR2 and HCDR3 comprising the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively and an immunoglobulin light chain LCDR1, LCDR2 and LCDR3 comprising the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively.
41. The assay method of claim 36, wherein the immunoglobulin heavy chain comprising the polypeptide sequence of SEQ ID NO: 8 and the immunoglobulin light chain comprises the polypeptide sequence of SEQ ID NO: 9.
42. The assay method of claim 39, wherein the immunoprecipitation antibody is PT9.
43. The assay method of claim 36, wherein sulfite modification at (i) residue 184 or 185, (ii) residue 191, and (iii) residue 208 are detected.
44. The assay method of claim 43, wherein sulfite modification at (i) residue 184 or 185, (ii) residue 191 , (iii) residue 208, and (iv) residue 400 are detected.
69
RECTIFIED SHEET (RULE 91 ) ISA/EP
45. The assay method of claim 36, wherein the liquid chromatography mass spectrometry is nanoflow liquid chromatography - high resolution mass spectrometry (nLC-HRMS), liquid chromatography - tandem mass spectrometry (LC-MS/MS), or nanoflow liquid chromatography - tandem mass spectrometry (nLC-MS/MS).
46. The assay method of claim 43, wherein sulfite modification at (i) residue 184 or 185 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 15 or 16.
47. The assay method of claim 43, wherein sulfite modification at (ii) residue 191 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 17.
48. The assay method of claim 43, wherein sulfite modification at (iii) residue 208 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 18.
49. The assay method of claim 44, wherein (9-GlcNAcylation at (iv) residue 400 is detected by detecting presence of a polypeptide having a sequence of SEQ ID NO: 19.
50. The method of any one of claims 1-8 and 10-12, wherein the amount of (9-GlnN Acylated tau peptides in the biological sample is determined using the assay methods of any one of claims 14-49.
51. The method of any one of claims 1-13 and 50, wherein the subject is a mammal.
52. The method of claim 51, wherein the subject is a mouse or a human.
70
RECTIFIED SHEET (RULE 91 ) ISA/EP
PCT/EP2023/073945 2022-08-31 2023-08-31 Mass spectroscopy assay for detecting o-beta-linked n-acetylglucosaminylated tau peptides WO2024047185A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263374132P 2022-08-31 2022-08-31
US63/374,132 2022-08-31

Publications (1)

Publication Number Publication Date
WO2024047185A1 true WO2024047185A1 (en) 2024-03-07

Family

ID=87974757

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/073945 WO2024047185A1 (en) 2022-08-31 2023-08-31 Mass spectroscopy assay for detecting o-beta-linked n-acetylglucosaminylated tau peptides

Country Status (1)

Country Link
WO (1) WO2024047185A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9879001B2 (en) * 2013-03-14 2018-01-30 Merck Patent Gmbh Glycosidase inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9879001B2 (en) * 2013-03-14 2018-01-30 Merck Patent Gmbh Glycosidase inhibitors

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
"Uniprot", Database accession no. P10636-8
ABHINANDANMARTIN, MOL IMMUNOL., vol. 45, 2008, pages 3832 - 9
ALMAGRO, MOLRECOGNIT, vol. 17, 2004, pages 132 - 43
ARNOLD C. SHANE ET AL: "The Microtubule-associated Protein Tau Is Extensively Modified with O-linked N-acetylglucosamine", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 46, 15 November 1996 (1996-11-15), US, pages 28741 - 28744, XP093110558, ISSN: 0021-9258, DOI: 10.1074/jbc.271.46.28741 *
BARTOLOME-NEBREDA ET AL.: "O-GlcNAcase inhibitors as potential therapeutics for the treatment of Alzheimer's disease and related taupathies: analysis of the patent literature", EXPERT OPINION ON THERAPEUTIC PATENTS, vol. 31, no. 12, 2021, pages 1117 - 1154
BIJTTEBIER SEBASTIAAN ET AL: "IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau", JOURNAL OF PROTEOME RESEARCH, vol. 22, no. 4, 8 March 2023 (2023-03-08), pages 1309 - 1321, XP093108043, DOI: 10.17632/8n5j45dnd8.1;10.17632/h5vdrx4n3d.1 *
BUTNERKIRSCHNER, J CELL BIOL., vol. 115, no. 3, 1991, pages 717 - 30
CALLE ET AL., J. AM. SOC. MASS SPECTROM., vol. 32, 2021, pages 2366 - 2375
CANTRELLE ET AL., FRONTIERS IN MOLECULAR NEUROSCIENCE, vol. 14, 2021, pages 661368
CHOTHIA ET AL., J. MOL. BIOL., vol. 227, 1992, pages 799 - 817
CHOTHIALESK, J MOLBIOL., vol. 196, 1987, pages 901 - 17
HANGER ET AL., TRENDS MOL MED., vol. 15, 2009, pages 112 - 9
IQBAL ET AL., CURR ALZHEIMER RES., vol. 7, no. 8, 2010, pages 656 - 664
KIM, MOLECULES, vol. 16, 2011, pages 1987 - 2022
LEFRANC ET AL., DEV COMP IMMUNOL., vol. 27, 2003, pages 55 - 77
MA XIAOFENG ET AL: "The emerging link between O-GlcNAcylation and neurological disorders", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, BIRKHAUSER VERLAG, HEIDELBERG, DE, vol. 74, no. 20, 22 May 2017 (2017-05-22), pages 3667 - 3686, XP036318281, ISSN: 1420-682X, [retrieved on 20170522], DOI: 10.1007/S00018-017-2542-9 *
MORRIS ET AL., NEURON, vol. 70, 2011, pages 410 - 26
SATO ET AL., NEURON, vol. 97, 2018, pages 1284 - 1298
SCOTT A YUZWA ET AL: "Mapping-GlcNAc modification sites on tau and generation of a site-specific-GlcNAc tau antibody", AMINO ACIDS ; THE FORUM FOR AMINO ACID AND PROTEIN RESEARCH, SPRINGER-VERLAG, VI, vol. 40, no. 3, 13 August 2010 (2010-08-13), pages 857 - 868, XP019883381, ISSN: 1438-2199, DOI: 10.1007/S00726-010-0705-1 *
TRAMONTANO ET AL., J. MOL. BIOL., vol. 215, 1990, pages 175 - 182
VANDERMEEREN MARC ET AL: "Anti-Tau Monoclonal Antibodies Derived from Soluble and Filamentous Tau Show Diverse Functional Properties in vitro and in vivo", JOURNAL OF ALZHEIMER`S DISEASE, vol. 65, no. 1, 7 August 2018 (2018-08-07), NL, pages 265 - 281, XP055860147, ISSN: 1387-2877, DOI: 10.3233/JAD-180404 *
WANG Z ET AL: "Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry", MOLECULAR & CELLULAR PROTEOMICS, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 9, no. 1, 1 January 2010 (2010-01-01), pages 153 - 160, XP002735035, ISSN: 1535-9476, [retrieved on 20090819], DOI: 10.1074/MCP.M900268-MCP200 *
WANG ZIHAO ET AL: "Site-Specific GlcNAcylation of Human Erythrocyte Proteins", DIABETES, vol. 58, no. 2, 1 February 2009 (2009-02-01), US, pages 309 - 317, XP093110141, ISSN: 0012-1797, DOI: 10.2337/db08-0994 *
WUKABAT, J EXP MED., vol. 132, 1970, pages 211 - 50

Similar Documents

Publication Publication Date Title
KR101826449B1 (en) Method for preparing peptide fragments, kit for preparing peptide fragments to be used therein, and analysis method
JP7399096B2 (en) Assays to detect neurodegeneration
KR102425339B1 (en) Method for measurement of vitamin d
KR20120100982A (en) Multiplex quantitation of individual recombinant proteins in a mixture by signature peptides and mass spectrometry
Mukherjee et al. Quantitative proteomics of tau and Aβ in detergent fractions from Alzheimer's disease brains
JP7215903B2 (en) Protein structural type detection
WO2024047185A1 (en) Mass spectroscopy assay for detecting o-beta-linked n-acetylglucosaminylated tau peptides
JPWO2019167830A1 (en) Antibodies that specifically recognize the N-terminus of APP669-x, and immunoassays
JP2024507755A (en) Method for detecting neurofilament light chains in plasma and cerebrospinal fluid
JP2016114573A (en) Pretreatment method of sample for oxytocin detection
JP2023533806A (en) A blood-based assay for detecting tauopathies or amyloidogenic diseases
CN115298215A (en) Specifically binds to primary immunodeficiency: antibodies to peptides related to viskott-aldrich syndrome and X-linked agammaglobulinemia
Bijttebier et al. IP-LC-MSMS enables identification of three tau O-GlcNAcylation sites as O-GlcNAcase inhibition pharmacodynamic readout in transgenic mice overexpressing human tau
JP6152908B2 (en) Method for preparing and analyzing peptide fragment
US11906530B2 (en) Methods for the detection of tau protein aggregates
EA045805B1 (en) TESTS FOR DETECTING NEURODEGENERATIVE DISEASES
KR20200067193A (en) Copy protection against antibodies
WO2005063335A2 (en) MONOCLONAL DES-LYS58-β2-MICROGLOBULIN ANTIBODIES FOR MEASUREMENT AND MANAGEMENT OF HEMODIALYSIS COMPLICATIONS
Maftei Molecular characterization of neuroprotective β-amyloid interacting peptides and autoantibodies relevant to Alzheimer’s disease
WO2012175201A1 (en) Methods for the characterization of antibodies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23767820

Country of ref document: EP

Kind code of ref document: A1