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US-20260126454-A1 - MASS SPECTROMETRY ANALYSIS OF MARKERS FOR ALZHEIMER'S DISEASE

US20260126454A1US 20260126454 A1US20260126454 A1US 20260126454A1US-20260126454-A1

Abstract

Provided herein is a sensitive, quantitative, and scalable targeted proteomics assay of Alzheimer's Disease biomarkers representing neuronal, glial, vasculature and metabolic pathways. The biomarkers are protease-digested peptides selected from biological samples of individuals having normal Aβ and Tau levels (AT−) and from symptomatic and asymptomatic individuals having low Aβ and high Tau levels (AT+). The assay uses selective reaction monitoring-based mass spectrometry (SRM-MS) of peptides in the biological samples after digestion.

Inventors

  • Nicholas Seyfried
  • Duc Duong
  • Allan Levey
  • James Lah
  • Caroline Watson

Assignees

  • EMORY UNIVERSITY

Dates

Publication Date
20260507
Application Date
20230825

Claims (20)

  1. 1 . A method for measuring multiple peptides indicative of cognitive function in a cerebrospinal fluid or plasma sample from a subject comprising (a) treating the cerebrospinal fluid or plasma sample from the subject with trypsin to produce a peptide solution comprising multiple peptides indicative of cognitive function, wherein the multiple peptides indicative of cognitive function comprise two or more of the peptides having the amino acid sequence of SEQ ID NO:1-53 and SEQ ID NO:69-116; (b) adding to the peptide solution a reference standard comprising isotopically labeled peptides to produce a test solution; (c) detecting the multiple peptides indicative of cognitive function and the isotopically labeled peptides in the test solution using selective reaction monitoring-based mass spectrometry; and (d) determining an amount of the multiple peptides indicative of cognitive function.
  2. 2 . The method of claim 1 , wherein the multiple peptides indicative of cognitive function comprise two or more peptides selected from the group consisting of (SEQ ID NO: 18) AAFNSGK, (SEQ ID NO: 22) AGALNSNDAFVLK:, (SEQ ID NO: 35) ALVILAK, (SEQ ID NO: 44) AQALEQAK; (SEQ ID NO: 20) DHLLGVSDSGK, (SEQ ID NO: 7) EAFSLFDK, (SEQ ID NO: 31) ELDVLQGR, (SEQ ID NO: 48) EPVAGDAVPGPK, (SEQ ID NO: 49) GLQEAAEER, (SEQ ID NO: 24) GQLSFNLR, (SEQ ID NO: 9) IASNTQSR, (SEQ ID NO: 18) IEEELGSK, (SEQ ID NO: 43) IESQTQEEVR, (SEQ ID NO: 32) LAIGFSTVQK, (SEQ ID NO: 33) LEGNPIVLGK, (SEQ ID NO: 39) LFEELVR, (SEQ ID NO: 15) LNVTEQEK, (SEQ ID NO: 51) NLLSVAYK, (SEQ ID NO: 46) QETLPSK, (SEQ ID NO: 3) QSELSAK, (SEQ ID NO: 30) VAELEDEK, (SEQ ID NO: 52) VISSIEQK, (SEQ ID NO: 19) YDNSLK, and (SEQ ID NO: 53) VVSSIEQK.
  3. 3 . The method of claim 2 , wherein the multiple peptides indicative of cognitive function comprise VISSIEQK (SEQ ID NO:52), VVSSIEQK (SEQ ID NO:53), and NLLSVAYK (SEQ ID NO:51).
  4. 4 . The method of claim 1 , wherein the multiple peptides indicative of cognitive function comprise two or more peptides selected from the group consisting of (SEQ ID NO: 69) AAQEEYVK, (SEQ ID NO: 70) ADQDTIR, (SEQ ID NO: 71) DGADFAK, (SEQ ID NO: 72) DGNGYISAAELR, (SEQ ID NO: 73) DIEEGAIVNPGR, (SEQ ID NO: 74) DYSVTANSK, (SEQ ID NO: 75) EGDCPVQSGK, (SEQ ID NO: 76) EHAVEGDCDFQLLK, (SEQ ID NO: 77) ELSDIAHR, (SEQ ID NO: 78) ENFSCLTR, (SEQ ID NO: 79) EPCGGLEDAVNEAK, (SEQ ID NO: 80) ESLSSYWESAK, (SEQ ID NO: 81) EVTGIITQGAR, (SEQ ID NO: 82) FIVYSYK, (SEQ ID NO: 83) FVEGLPINDFSR, (SEQ ID NO: 84) GALQNIIPASTGAAK, (SEQ ID NO: 85) GDLGIEIPAEK, (SEQ ID NO: 86) GDSVVYGLR, (SEQ ID NO: 87) GDYPLEAVR, (SEQ ID NO: 88) GECVPGEQEPEPILIPR, (SEQ ID NO: 89) GNDISSGTVLSDYVGSGPPK, (SEQ ID NO: 90) GNQWVGYDDQESVK, (SEQ ID NO: 91) GVCEETSGAYEK, (SEQ ID NO: 92) GVNLPGAAVDLPAVSEK, (SEQ ID NO: 93) HVLFGTVGVPEHTYR, (SEQ ID NO: 94) HYGGLTGLNK, (SEQ ID NO: 95) ICEPGYSPTYK, (SEQ ID NO: 96) IVFLEEASQQEK, (SEQ ID NO: 97) LIVHNGYCDGR, (SEQ ID NO: 98) LLVFATDDGFHFAGDGK, (SEQ ID NO: 99) LYEQLSGK, (SEQ ID NO: 100) SGQLGIQEEDLR, (SEQ ID NO: 101) TATSEYQTFFNPR, (SEQ ID NO: 102) TEAADLCK, (SEQ ID NO: 103) TLLSVGGWNFGSQR, (SEQ ID NO: 104) VFEDESGK, (SEQ ID NO: 105) VGNLTVVGK, (SEQ ID NO: 106) VIGSGCNLDSAR, (SEQ ID NO: 107) VIVVGNPANTNCLTASK, (SEQ ID NO: 108) VNQIGSVTEAIQACK, (SEQ ID NO: 109) VTLSAAPPSYFR, (SEQ ID NO: 110) VVEGSFVYK, (SEQ ID NO: 111) WGLGGTCVNVGCIPK, (SEQ ID NO: 112) YGFIEGHVVIPR, (SEQ ID NO: 113) YISPDQLADLYK, (SEQ ID NO: 114) YLAEVATGEK, (SEQ ID NO: 115) YLIPNATQPESK, and (SEQ ID NO: 116) YVWLVYEQDRPLK.
  5. 5 . The method of claim 1 , wherein the isotopically labeled peptides comprise peptides having the amino acid sequences of SEQ ID NO:63-68 with labeled C-terminal lysine or arginine residues.
  6. 6 . The method of claim 1 , further comprising identifying an Alzheimer's Disease state in the subject.
  7. 7 . The method of claim 6 , wherein the Alzheimer's disease state of the subject is Alzheimer's Disease positive or Alzheimer's Disease negative.
  8. 8 . The method of claim 7 , wherein the detected peptides indicative of Alzheimer's Disease positive state comprise peptide fragments of glucose metabolism enzymes.
  9. 9 . The method of claim 6 , wherein the glucose metabolism enzymes comprise PKM, MDH1, ENO1, ALDOA, ENO2, LDHB, and TPI1.
  10. 10 . The method of claim 7 , wherein the detected peptides indicating the subject is Alzheimer's Disease positive comprise; a) two or more of the peptides having the amino acid sequence of SEQ ID NO:44, SEQ ID NO:20, SEQ ID NO:39, SEQ ID NO:57, SEQ ID NO:55, SEQ ID NO:51, SEQ ID NO:53 SEQ ID NO:52, or SEQ ID NO:19; b) SEQ ID NO:53, SEQ ID NO: 51, SEQ ID NO:44, SEQ ID NO:52, and SEQ ID NO:19; or c) SEQ ID NO: 96 and SEQ ID NO: 115.
  11. 11 - 12 . (canceled)
  12. 13 . The method of claim 6 , wherein the Alzheimer's Disease state of the subject is asymptomatic Alzheimer's Disease.
  13. 14 . The method of claim 13 , wherein the detected peptides indicative of asymptomatic Alzheimer's Disease state comprise: a) two or more peptides having the amino acid sequence of SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:20, SEQ ID NO:51, or SEQ ID NO:53; b) peptides having the amino acid sequence of SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:20, SEQ ID NO:51, or SEQ ID NO:53; or c) SEQ ID NO: 96 and SEQ ID NO: 115.
  14. 15 - 16 . (canceled)
  15. 17 . The method of claim 6 , wherein the Alzheimer's Disease state of the subject is symptomatic Alzheimer's Disease.
  16. 18 . The method of claim 17 , wherein the detected peptides indicative of symptomatic Alzheimer's Disease state comprise: a) two or more peptides having the amino acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:48, SEQ ID NO:43, or SEQ ID NO:53; b) peptides having the amino acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:48, SEQ ID NO:43, or SEQ ID NO:53; or c) SEQ ID NO: 70 and SEQ ID NO: 100.
  17. 19 - 20 . (canceled)
  18. 21 . The method of claim 6 , wherein the Alzheimer's Disease state is further characterized as AT+ or AT−.
  19. 22 . The method of claim 6 , further comprising genotyping the subject positive for Alzheimer's Disease by detecting one or more peptides indicative of apolipoprotein E (APOE), albumin (ALB), hemoglobin subunit A (HBA), or hemoglobin subunit B (HBB) expression in the test solution, wherein detecting the one or more peptides indicative of APOE, ALB, HBA, or HBB expression is performed concurrently by selective reaction monitoring-based mass spectrometry.
  20. 23 . The method of claim 22 , wherein the method further comprises detecting one or more peptides indicative of APOE expression and wherein the APOE expression comprises expression of one or more of APOE2, APOE3, or APOE4.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This is the U.S. National Stage of International Application No. PCT/US2023/072936, filed Aug. 25, 2023, which was published in English under PCT Article 21 (2), which in turn claims the benefit of U.S. Provisional Application No. 63/401,551, filed on Aug. 26, 2022, the entire disclosure of each application is hereby incorporated herein by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. AG046161 and AG025688 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS XML VIA EFS-WEB The instant application contains a Sequence Listing that is filed electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on Feb. 21, 2025, is named 6975-112563-03_Sequence Listing.xml and is 100,730 bytes in size. BACKGROUND Alzheimer's disease (AD) is the most common form of dementia, affecting more than 45 million people worldwide. Cerebrospinal fluid (CSF) β-amyloid (Aβ), Tau, and phosphorylated Tau currently provide the most sensitive and specific biomarkers for diagnosis. However, these diagnostic biomarkers do not reflect the complex changes in AD brains beyond Aβ plaques and Tau neurofibrillary tangles (NFT) and thus fail to reflect the heterogeneous and complex changes associated with the disease. Failed clinical trials in the treatment of AD highlight the need for advancements in diagnostic profiling, disease monitoring, and treatment evaluation. SUMMARY Provided herein is a sensitive, quantitative, and scalable targeted proteomics assay of AD biomarkers representing neuronal, glial, vasculature and metabolic pathways. The biomarkers are protease-digested peptides selected from biological samples of individuals having normal Aβ and Tau levels (AT−) and from symptomatic and asymptomatic individuals having low Aβ and high Tau levels (AT+). The assay uses selective reaction monitoring-based mass spectrometry (SRM-MS) of peptides in the biological samples after digestion. Isotopically labeled peptide standards are added as internal standards for relative quantification. Thus, provided herein is a method for measuring multiple peptides indicative of cognitive function in a biological sample (e.g., a cerebrospinal fluid or plasma sample) from a subject. The method includes treating the sample from the subject with a protease to produce a peptide solution comprising multiple peptides indicative of cognitive function, wherein the multiple peptides indicative of cognitive function comprise two or more of the peptides having SEQ ID NO:1-53 and SEQ ID NO:69-116; adding to the peptide solution a reference standard comprising isotopically labeled peptides to produce a test solution; detecting the multiple peptides indicative of cognitive function and the isotopically labeled peptides in the test solution using selective reaction monitoring-based mass spectrometry; and determining an amount of the multiple peptides indicative of cognitive function. The method permits determining the Alzheimer's disease state (e.g., positive/negative, asymptomatic/symptomatic, and mild cognitive impairment/early-stage AD/late-stage AD). Also provided herein are methods of treating a subject with or at risk of developing AD. The method comprises utilizing the method for measuring multiple peptides indicative of cognitive function in a biological sample from a subject and administering a treatment (e.g., a therapeutic agent) to the subject. Optionally the method for measuring multiple peptides indicative of cognitive function in a biological sample to detect changes in brain function and efficacy of treatment. A kit comprising one or more reagents for performing the method of measuring multiple peptides indicative of cognitive function in a biological sample is also provided. The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1F shows cohort characteristics. A total of 390 samples (133 controls, 127 asymptomatic AD (AsymAD), and 130 AD unless otherwise noted) were analyzed using the identified cohort characteristics for grouping. FIG. 1A shows the age range across each group of the cohort, which were carefully selected to balance for age and sex (see also Table 2). FIG. 1B shows the range of cognition assessed using the Montreal Cognitive Assessment (MoCA) score. There is no significant difference in scores between the Control and AsymAD groups serving as the two cognitively normal diagnostic groups; however, a significant decrease in cognition scores was observed between the controls and AsymAD and the controls and AD group (133 controls, 127 AsymAD, 124 AD). FIG. 1C shows the Roche Diagnostics E