WO2015191825A1

WO2015191825A1 - Methods for the detection and measurement of amyloid beta in biological samples

Info

Publication number: WO2015191825A1
Application number: PCT/US2015/035282
Authority: WO
Inventors: Chaoran Ron HUANG; Tao Ye; Liyu Yang
Original assignee: Biogen Ma Inc.
Priority date: 2014-06-13
Filing date: 2015-06-11
Publication date: 2015-12-17

Abstract

Methods for detecting and measuring pΕ3-Αβ and uses of the methods to diagnose and/or prognose diseases associated with altered levels of pΕ3-Αβ are disclosed. Uses of the methods to develop., tailor, and direct therapies are also disclosed.

Description

METHODS FOR THE DETECTION AND MEASUREMENT OF AMYLOID BETA IN BIOLOGICAL SAMPLES

[001] This application claims the benefit of U.S. Provisional Application No. 62/01 1 ,960, filed June 1 .3, 2014, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[Θ02] The disclosure relates to methods for detecting and measuring amyloid β in biological samples and diagnostic, prognostic, and therapeutic uses thereof. In a particular aspect, the disclosure relates to highly sensitive multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/ S) methods for detecting N- terminal truncated amyloid β, e.g., pyrogiutamate amyloid β.

[003] Peptides that are normally soluble can aggregate into insoluble configurations, such as amyloid and amyloid-like structures, that contribute to disease. Amyloidosis refers to a diverse group of diseases (e.g., Alzheimer's disease, Down syndrome, type 2 diabetes) characterized by the deposition of insoluble amyloids (Blancas-Mejia and Ramirez-Alvarado, 2013: Hazenberg 2013). As these amyloids continue to accumulate, they can interfere with the normal functions of cells, tissues, and organs (Blancas-Mejia and Ramirez-Alvarado. 2013; Hazenberg 2013; Haass and Selkoe, 2007). Although the precursor molecules for these diseases differ, the amyloid assreeates in each disease share similar structural and functional features. Amyloid aggregates can become insoluble, toxic, and important to disease progression (Blancas- Mejia and Ramirez-Alvarado, 201 3; Hazenberg 2013: WO201 1076854: US8512677).

[004] In particular, amyloid β (Αβ) is thought to play an important role in the development of Alzheimer's disease (AD) (Haass and Selkoe, 2007). AD is a neurodegenerative disease characterized by Αβ plaque formation, neuron death, and debilitating memory loss. Disease progression is relativeiy slow, and the first symptoms of AD only manifest after several decades of neuron loss (Blennow et aL, 2006), At present, AD is impossible to treat and extremely difficult to diagnose in early stages.

[005] Αβ plaques i the AD brain comprise Αβ oligomers (Haass and Selkoe. 2007), and understanding Αβ accumulation is believed to be critical for the development of early diagnostic tests and eventual therapeutic agents for AD. Αβ is derived from the proteolytic cleavage of amyloid precursor protein (APP). APP can be cleaved at multiple sites and into fragments of many different lengths. Thus, Αβ in the brain is not a single distinct species, but rather a heterogeneous collection of peptide species, e.g., 30-43 amino acids in length, with different C- and N-terminal sequences (Gunn et aL, 2010; Haass and Selkoe, 2007), These different species vary in toxicity and aggregation propensity,

[§06] Pyroglutamate Αβ (ρΕ-Αβ), and in particular ρΕ3-Αβ, is recognized in the art as one of the most toxic and aggregation-prone forms of Αβ (Mori et aL, 1992; Russo et aL, 2002; Schilling et aL, 2006: Acero et aL, 2009: Wirths et al._s 2009; Jawhar et a!., 201 1), ρΕ3-Αβ is formed by a multi-step process that begins with the removal of the first two amino acids of Αβ to expose a glutamate at position three. The enzyme glutamray] cyclase then catalyzes pyroglutamate formation by the dehydration and cyclization of glutamate (Schilling et aL, 2004). ρΕ3-Αβ exhibits increased

hydrophobicity and stability and is particularly resistant to degradation by peptidases compared to unmodified Αβ (Saido et aL, 1996; Jawhar et ah, 201 1).

[007] As a result, ρΕ3-Αβ exhibits "up to 250-fold accelerated initial formation of aggregates compared to unmodified Αβ" and 'is crucial for the initiation of disease'^" (Schilling et aL, 2006). Not only does ρΕ3-Αβ precede the deposition of unmodified Αβ in the brain, ρΕ3-Αβ exceeds the deposition of unmodified Αβ and "may account for more than 50% of Ap accumulated in plaques" (Russo et al„, 2002). ρΕ3-Αβ is considered to be a promising target for AD diagnosis, prognosis, and therapy (DeMattos et aL, 201 1 ), Treating diseased neurons in vitro with antibodies to ρΕ3-Αβ reduces toxicity, and in mouse models, "reduce[s] Αβ plaque load and normalizejs] behavioral deficits" (Frost et aL 2012: Wirths et a!., 2010; WO2011 151076: US8283517).

[008] In addition, increased C -terminal length (e.g., the Αβ_χ-42 species compared to the Αβ_χ-4ο species) and/or decreased N-ierminal length (e.g., the Αβ_χ.42 species compared to the Apj.₄₂ species) enhance aggregation and promote toxicity in both ρΕ3-Αβ and unmodified Αβ peptides (Jawhar et aL, 201 1 ). For example, Αβ_ρο...¾2 precedes the deposition of unmodified Αβ;,_χ in the brain and is more toxic than unmodified Αβ in AD and Down syndrome (Lemere et aL, 1996; Russo et aL, 2002). n transgenic mice, expression of Αβ_ρο· 2 causes "massive neurological impairments" starting in early adulthood (Wirths et aL. 2009). By contrast, transgenic mice that only express unmodified Αβ exhibit relatively "minimal neurofibrillary pathology and neuronal loss" (Kawarabayashi et aL, 2001).

[009] Although ρΕ3-Αβ demonstrates tremendous diagnostic, prognostic, and therapeutic potential, detecting and measuring ρΕ3-Αβ currently poses many technical challenges. For example, current methods are able to detect ρΕ3-Αβ primarily in biological samples containing relatively high levels of ρΕ3-Αβ, such as Αβ plaques and post-mortem AD brains, but detection remains challenging in samples containing relatively low levels of ρΕ3-Αβ, such as cerebrospinal fluid. For example, ρΕ3-Αβ has been detected in plaques from AD and Down syndrome patients (Frost et aL, 2013) and AD mice ( WO2012021469) by imraunohistochemistry. Several antibodies have been developed to detect ρΕ3-Αβ in post-mortem brain tissue from: AD patients by immunohistochernistry (De Kimpe et a!., 2013; WO2.01 1 1 51076); AD patients by ELISA ( orawski et al., 2013); AD mice by EL1SA ( Wu et al., 2013: U S8058405); AD mice by immunohi stochemi stry (US8512677); AD patients, AD mice, and aged rhesus macaques by imraunohistochemistTy (Wirths et ai., 2013); AD patients by sandwich ELISA (Wirths et ah, 2010); and AD patients at different disease stages by

immunohistochemistry (Mandler et ah, 2012). ρΕ3-Αβ has been detected in plasma from AD patients by sandwich ELISA (WO201 1 151076). However, enhancing specificity, selectivity, and sensitivity, in particular for quantitative assays, may require analytical methods such as mass spectrometry and other technologies.

[010] ΑβρΕΜο, ΑβρΕ3-42, Αβι.42, Α _{5 -}4ο, Αβ₄^2, and Aps- 2 have been detected in murine AD brains by immimopreeipitation combined with mass spectrometry (Wittnam et ah, 2012). Αβ). ο, Αβι^, Αβ) .₃9, β^, Αβ· _-37, Αβ₂-4ϋ₅ Αβ_3-40·, ^ ά ρΕ3-Αβ have been detected in murine AD brains by one- and two-dimensional gel electrophoresis combined with immunobiotting and mass spectrometry (Bibl et ai., 2012). Αβ_ΡΕ3-42» Αβι-42, Αβ/:.₄₂, and Αβ_{ί -4}ο have been detected in brain tissues from patients with AD, sporadic Alzheimer's disease (SAD), and familial Alzheimer's dementias (FAD) by immunoprecipitation combined with mass spectrometry (Portelius et al, 2010),

[0.1.1] in AD, Αβ plaque formation may be associated with altered levels of Αβ, ρΕ~Αβ, and/or ρΕ3 ~Αβ, and the detection and/or q antitation of these biornarkers may be useful for early detection and diagnosis of AD, Early detection is especially critical for therapeutic intervention, as neuron loss begins several decades before the onset of AD symptoms (Eilennow et ah, 2006). In particular, cerebrospinal fluid (CSF) provides a more rapid and sensitive readout of changes in the brain conipared to other biological samples, and it is known that AD even in its earliest stages can cause changes in CSF (Blennow et ah, 2009; Mehta et ah, 2000; Blennow and Hampel, 2003). Thus, a highly sensitive method for detecting and measuring ρΕ3-Αβ in CSF may allow not only early detection of disease, but may provide an accessible biological sample ior use in drag development, a process for which it is crucial to identify and monitor the effects of drugs in patients. However, CSF contains particularly low concentrations of Αβ, especially in AD patients { Mehta et ah, 2000; Kavvarabayaslii et ah, 2001 ; Biennow and Hampel, 2003: Fagan et al, 2006; Strozyk et al., 2003), which poses a significant technical challenge for detection. Thus, there is a significant unmet need for highly sensitive assays for detecting and measuring ρΕ3-Αβ, particularly for biological samples containing low amounts or concentrations of ρΕ3-Αβ₅ e.g.. cerebrospinal fluid.

[012] ^'The invention provides methods for detecting and measuring amyloid β in biological samples and diagnostic, prognostic, and therapeutic uses thereof. In specific embodiments, the invention provides highly sensitive and reliable methods for detecting and measuring ρΕ3-Αβ by LC-MS/MS. The methods of the invention may be used to detect and measure ρΕ3-Αβ in biological samples containing low levels of pE3~ Αβ, such as samples that are very dilute and/or samples of very limited volume. In particular, the methods of the invention described herein may be used to detect and measure ρΕ3-Αβ in cerebrospinal fluid, in further embodiments, the methods of the invention may be used to distinguish between ρΕ3-Αβ peptides of different lengths.

[013] In some embodiments, the meihods of the invention are used to diagnose and prognose diseases associated with altered levels of ρΕ3-Αβ. In certain

embodiments, the disease is AD, in particular the early stages of At), in further embodiments, the methods of the invention may be used to stratify disease slates, monitor disease progression, and/or assess responsiveness to treatment. In addition, the methods of the invention may be used to tailor and direct therapies. BRIEF DESCRIPTION OF THE FIGURES

[014] FIG. 1 shows a schematic diagram of the steps involved in one embodiment of the invention, A biological sainpie is prepared and undergoes liquid chromatography (LC) separation. The sample is subjected to atmospheric pressure ionization, selected and fragmented according to mass-to-charge ratio, and detected by tandem mass spectrometry (MS/MS).

[015] FIG, 2 shows a mass spectrum of an Αβ_ΡΕ3- 0 full scan.

[016] FIG, 3 shows a mass spectrum of an Αβ_ΡΕ3- ο M+4H product ion scan.

[017] FIG, 4 shows standard curves of A β - .38, Αβι-40, Αβ·^, Αβ,,^ο, Αβ Ε3- ο, and ΑβρΕ3-42 using solid-phase extraction. A calibration curve of 10 pg/niL - 8 ng/mL is achieved,

[018] FIGS. 5A-C show various Αβ species that were detected in human CSF. Apj . , Αβι-4ο, Αβ| .33 were detected in pooled human CSF without using solid-phase extraction (FIG. 5 A). Αβ_ΡΕ3-3ο and Αβ_ΡΕ3-42 were below the limit of detection (LOD) and Αβ_ΡΕ3-4ο below the limit of quantitation (LOQ) in pooled human CSF after using solid-phase extraction (FIG. SB) and in pooled human CS F with 1 0 pg/m L Αβ

(comprising Αβι_ 2, Αβ( . ο, Αβ ι Αβ_ΡΕ3- ο, Αβ_ΡΕ3-3ο, and Αβ_ΡΕ3- 2) after using solid- phase extraction (FIG, SC).

[01 ] FIG. 6 show lysyi endopeptidase (Lys-C) cleavage at the C-terrninus of a lysine residue (K), which is located at position 16 of Αβ, for Αβ_Ρ 3-42, Αβι. 2, amyloid precursor protein.

[020] FIG, 7 shows exemplary chromatograms of 20 pg/mL or 2 pg mL of Αβ_Ρ£3.ΐ6 and Αβι .._{) 6} purified by using Lys-C digestion and solid-phase extraction.

[021] FIG, 8 shows immunoprecipitation optimization using 150 μΐ, of 4 pg/mL - 2 ng/mL of Αβ_ΡΕ3.]₆. [022] FIG, 9 shows immunoprecipitation optimization using 2,4 pg mL - 9,6

[023] FIG. i O shows the sensitivity and specificity for particular cutoff concentrations of pE3-Aj3 in CSF samples.

[024] FIG. 11. demonstrates that the concentration of ρΕ3-Αβ in the CvSF of AD patients is significantly lower than the concentration of ρΕ3~Αβ in the CSF of control patients,

DESCRIPTION OF EMBODI MENTS

[025] In order that the disclosure may be more readily understood, certain terms are first defined. T hese definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[026] The term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass-to-charge ratio, or rn/z. MS is used broadly to include all components and systems that may be used to detect and identify analyies using their mass-to-charge ratio. The terms "analyte," "sample," "material,"

''chemical/' and "ion" may all be used herein to refer to a substance to be analyzed and identified. MS technology generally includes ionizing the compounds to form charged compounds, detecting the molecular weight of the charged compounds, and calculating the raass-to-charge ratio. The compounds may be ionized and detected by any suitable means.

1027] A "mass spectrometer" generally includes an ionizer and an ion detector.: In general, an analyte of interest is ionized, and the ions are subsequently introduced to magnetic and/or electric fields where the Ions follow a path that is dependent upon mass and charge. Examples of mass spectrometers include, but are not limited to. Waters Xevo TQ-S, ABSciex API 5500, and ABSciex API 4000.

[028] "Tandem mass spectrometry" or "MS/MS" refers to mass spectrometry in which multiple stages of mass analysis are performed, wherein the multiple stages are separated by time or by space. For example, tandem mass spectrometry in time can involve one mass analyzer (e.g., an ion trap), in which particular ions are first isolated, trapped, and fragmented, then analyzed by the same mass analyzer. Tandem mass spectrometry in space can involve more than one analyzer. The analyzers are separated, e.g., by a collision cell in which a gas (e.g., argon, xenon, nitrogen, helium) collides with the selected sample ions to bring about fragmentation. In general, two analyzers are used and the analyzers may be of the same or different types.

[029] "ionization" or "ionizing" refers to a process by which an analyte ion having a net electrical charge equal to one or more electron units is generated. Negative ions have a net negative charge of one or more electron units, and positive ions have a net positive charge of one or more electron units.

[030] 'Tdectrospray ionization" or "ESI" refers to an ionization method in which an analyte of interest is subjected to desolvation, wherein a sample solution containing the analyte of interest is sprayed into an electric field to form charged droplets.

[031] "Atmospheric pressure chemical ionization" or "APCI" refers to an ionization method that produces ions by ion-molecule interactions that occur at atmospheric pressure.

[032] "Solid-phase extraction" or "SPE" refers to a process by which a mixture is separated into components. The components are dissolved and/or suspended in solution ("mobile phase") and exhibit different affinities for a solid through which the solution is passed ("solid phase"). In some instances, as the mobile phase passes through the solid phase, undesired components of the mobile phase may be retained by the solid phase (i.e., the anaiyte in the mobile phase is purified), in oilier instances, desired components may be retained by the solid phase (i.e., the anaiyte of interest is retained in the solid phase), and a second mobile phase is used to eiute the retained anaiyte of the solid phase for further processing or analysis.

[033] "Mixed mode SPE" refers to an SPE technique that uses multiple retention mechanisms in the same column, For example, the solid phase of a mixed mode SPE column may exhibit strong cation exchange and hydrophobic retention.

[034] "Chromatography" refers to a process by which a mixture carried by a liquid or gas is separated into components that eiute at different retention times as a result of differential distribution of the chemical entities as they flow over a stationary liquid or solid phase,

[035] '^''Liquid chromatography" or "LC refers to the selective impedance of one or more components of a fluid solution as the fluid moves through a column. The impedance results from the distribution of the components of the mixture between one or more stationary phases and the mobile phase. Examples of LC include normal phase liquid chromatography (NPLC), reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance liquid

chromatography (UHPLC), high turbulence liquid, chromatography (HTLC), and multidimensional liquid chromatography. Examples of liquid chromatography instruments include, but are not limited to, the Waters UPLC i-Class and Shiamdzu LC- 20. [036] "High performance liquid chromatography," "high pressure liquid chromatography," and "HPLC" are used interchangeably to refer to liquid

chromatography in which the degree of separation is increased by forcing the mobile phase through a stationary phase (e.g., a densely packed column) under pressure.

[037] "Selective ion monitoring" is a detection mode for a mass spectrometer in which only ions within a relatively narrow mass range are detected.

[Θ38] "Multiple reaction monitoring" refers to a detection mode for a mass spectrometer in which a precursor ion and one or more fragment ions are selectively detected.

[039] "Limit of quantitation," "LOQ," "lo wer limit of quantitation," and "LLOQ" are used interchangeably to refer to a point at which measurements become quantitatively meaningful. In general, the LOQ is defined as the lowest concentration at which the measurement is reproducible within a relative standard deviation of +/- 20%.

{040] "Limit of detection" or "LOD" refers to a point at which the measured value is greater than the uncertainty associated with it. In general, the uncertainty is defined as three times the relative standard deviation from the mean signal intensity at zero concentration.

[041] "Purification" refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest. As used herein, the term "purification" does not refer to removing al l material from the sample other than the analyte(s) of interest.

|042| "Immunoprecipkation" or "IP" refers to a purification procedure that utilizes antibodies, including polyclonal or monoclonal antibodies, to enrich the one or more analytes of interest. Imraunopreci i tati on can be performed using any of the immunoprecipitation methods well known in the art. [043] "Amyloid β" and "Αβ" are used interchangeably to refer to amyloid β peptide and modifications, including pyrogkitamate Αβ, fragments, and equivalents thereof. In particular, "Αβ" as used herein refers to any fragment produced by the proteolytic cleavage of amyloid precursor protein (APP). "Αβ oligomers" is used to refer to multimeric species of Αβ that result from the association of monomeric Αβ species.

{044} The term "amyloidosis" refers to a group of diseases characterized by the deposition of insoluble amyloid plaques, including but not limited to, Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA) Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creuizfeld Jacob disease, Parkinson's disease, HIV-associated dementia (MAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, and secondary amyloidosis resulting from other diseases, including but not limited to, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.

[045] "ρΕ3-Αβ" or "Αβ_ρΕ as used herein refers to N-terrninally modified pyroglutamate amyloid β peptides that start at the glutamic acid (Glu) residue at position 3 of the amino acid sequence of Αβ, wherein the Glu residue is cycfized to form a pyroglutamic acid residue. Examples include but are not limited to Αβ_ΡΕ 8> Αβ Ε3·40> arcd Αβρπ_.3.42. The amino acid sequences of these N-terminally modified form s of Αβ peptides are:

AP 3-38 (SEQ ID NO: 1): Giu-Phe- \rg-His-Asp-Ser-Gly-Tyr-Glu-Val Jis-His-Gln ..ys-Leu-Va]-Phe- Phe-AIa-Glu-Asp-Val-GIy-Ser-Asn-Lys-C^y-Ala-Ile-Ile-Gly-I u-Met-Val-Gly-

Giy

Αβ 3-40 (SEQ ID NO: 2):

Phe-Ala-Giu-Asp-Val-Cay-Ser-Asn-Lys-Gly-Ala 1e-Ile--Gly-Leu~Met-Val-Gly-

Gly-Val-Vaj

Αβ 3-42 (SEQ ID NO: 3):

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-CHi^

Phe-Ala-Glu-Asp-Val-{:Hy-Ser-Asn .ys-Gly-Afa-Ile-ile~Gly-Leu-Mei-Val-Gly-

G iy- Val- Vai-ile- Ala

[046] "Unmodified Αβ'" is used to refer to amyloid β peptides in which the N terminus is not modified post-trans!aiional iy, including but not limited to Αβι.38, Ap 0> and Αβΐ j -42. The amino acid sequences of these unmodified Αβ peptides are:

Αβ 1 -38 (SEQ ID NO: 4):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-G 1 u-Val - His-Hi s-Gln- Lys-Leu-

Val-Phe-Phe-Ala-GIu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-l ie-Ile-Giy-Leu-Met-

Val-Gly-Gly

Αβ 1 -40 (SEQ ID NO: 5 ):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-ilis-Hls-Gln-Lys-Leu- Val-Phe-Phe-Ala-G3u-Asp-Vai-G3y-Ser-Asn-Lys-Gly-A3a-ile-lie-Gly-Leu-Met-

Val-Gly-Gly~Val-Va! Αβ 1 -42 (SEQ ID NO: 6):

Asp-A!a-G!u-Phe-Arg-His-Asp-Ser-Gly-Ty^

Val-Phe'Phe-Ala-Glu-AspA_' ^?al-Gly-Ser-Asn-Lys-Gly~Ala 1e 3e-G}y _ ^>u-rv]ei- Vai-GIy-Giy-Vai-Val-ile-Aia

[047] An antibody that is "specific for ρΕ3-Αβ" or a "pE3-AP-specific antibody" refers to an antibody that exhibits binding affinity for pE3-Ap and does not exhibit significant binding affinity for other forms of Αβ. including other forms of pyroglutamate Αβ, e.g., pEl l-Αβ. Examples of pE3 - Αβ-specific antibodies include but are not limited to:

AB 100-1 1 ;

9D5 (DSM ACC3Q56);

8C4 (DSM ACC3066);

AB 5-5-6 (DSM ACC2923);

AB 6-1 -6 (DSM ACC2924);

AB 17-4-3 (DSM ACC2925);

AB 24-2-3 (DSM ACC2926);

B12L, comprising the amino acid sequences of

SEQ ID NO; 7 (light chain):

DIVMTQI LSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPG QSPQLLiYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQW VDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEia-lKVYACEVTHQGLSSPVT SFNRGEC and SEQ ID NO: 8 (heavy chain):

QVQLVQSGAEVKKPGSSVKVSC ASGYDFT YY1N VRQAPGQG LEWMGWiNPGSGNT YNEKf GRVTITADESTSTAYMELSSLRSE DTAVYYCAREGITVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLYKDYFPEPVTVSWNSGA3.TSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYIC V^Ni-iKPSNT VDK VEP SCD Ti-iTC V VArV.] I Οϋ *νΉ W PKDTLMiSRTPEV i CVV v DYSi iKD E VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN G EY C VSNKALPAPiE TlSKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYK rPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALH HYTQKSLSLSPG; R17L, comprising the amino acid sequences of SEQ ID NO: 7 (light chain) and SEQ ID NO: 9 (heavy chain):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYYINWVRQAPGQG LEW GWINPGSGNTKY EKFKGRVTITADESTSTAYMELSSLRSE DTAVYYCAREGTTVYWGQG^'rrVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCI,VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNA TKPREEQYNSTTRVVSVLTVEHQDWEN GKEYKCKVS KALPAPIE ^'IIS AKGQPREF'QV YTEPPSRDELT N QVSLTCLV GFYPSDIAVEWESNGQPENYKTTPPVLDSDGSFFLY^'S LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

hE8L, comprising the amino acid sequences of SEQ ID NOs: 7 (light chain) and SEQ ID NO: 10 (heavy chain): QVQLVQSGAEVK PGSSVKVSC ASGYTFrDYYI WVRQAPGQG LEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSE DTAYY YCAREGETV YWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYiCNV H PSNT VDKKVEPKSCDKTI-ITC PPCPAPEEEGGPSVFLFPP PKDTL iSRTPEVTCVVVDVSHEDPE V FNWYV7)GVEVFr AKTKPREEQYNSTYT VVSVLTVLHQDWLN GKEYKC VSN ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPE NY TTPPVLDSDGSFFL YSKLTVDKSRWQQG VFSCS V MP⁾ EALH H YTQKSLS LSPG; CI-C7, comprising the arairso acid sequences of

SEQ ID NO: 11 (light chain):

D ί QMTQSPS SLS AS VGDRVTiTCKSTRS LEY S R.SKT YEN \V Y QQ P

GKAPKLLIYAVSKLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCVOO 11 lYS l F(i(f(;^'i KVEIK i^AAPSVHl-I 'Snf^M.KSGTASV

VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

STLTLS ADYEKHKVYACEVTHQGLSSPVmSFNRGEC and

SEQ ID NO: 12 (heavy chain):

EVQLVQSGAEVKKPGEiSLKlSCKGSGYTFTDYYINWVRQMPGKG

LEW GWiNPGSGNTKYNEKFKGQVTISADKSISTAYLQWSSEKA

SDTAMY YCAREGVTVYWGQGTLVTVSSASTKGPSVFPLAPSS S

TSGGTAALGCLVKDY PEPV^'rV^'SXVNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNH PSNT VDKKVEPKSCDKT

F]rcPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSl-IE DPEV FNWYVDGVEVH AKTKPREEQYNSTYR.VVSVLTVLHQE) WLNGKEYKCK^SNKALPAPiEKTISKAKGQPREPQVYTLPPSRDE LT NQVS LTC LVKGF YPSD1 AV EWES GQPEN YKTTPP VLDSD

GSFFLYS LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

G.

[048] "Polypeptide^'", ^'''pe tide¹', and "protein" are used interchangeably to refer to a polymer of amino acids.

[049] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

[05Θ] The phrase "and/or," when used, between elements in a list, is intended to mean either ( 1 ) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may he A alone; B alone; C alone: A and B; A and C; B and C; or A, B, and C. The phrase "and or" may be used interchangeably with "at least one of or "one or more of the elements in a list.

[051] The invention provides methods for detecting and measuring Αβ in biological samples. In particular, the invention provides highly sensitive and reliable multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for detecting and measuring ρΕ3-Αβ. The methods of the invention may be used for biological samples containing low levels of ρΕ3~Αβ such as samples that are very dilute and/or samples of very limited volume, in particular, the invention may be used to detect and measure ρΕ3~Αβ in cerebrospinal fluid.

[052J In some embodiments, a biological sample that potentially contains pF.3- Αβ is analyzed by mass spectrometry. In one aspect of these embodiments, the biological sample is first purified to remove some or al l of the interfering substances from biomatrices in preparation for mass spectrometry, The biological sample s then separated on the LC column and ionized by MS to generate anaiyte ions having a net electrical charge. Precursor Ions are filtered by mass-to-eharge ratio in a first mass filter (Q l). Anaiyte ions collide with gases in a collision cell (q2) in a collision-induced dissociation step, which further fragments the anaiyte ions. Fragment ions are then filtered by mass-to-charge ratio in a second mass filter (Q3). The fragment ions are detected, and the molecular weight and the mass-to-charge ratio are calculated. In alternate embodiments, fragment Ions are filtered by mass-to-eharge ratio and detected as electronic signals. In certain embodiments, the mass spectrometry Is tandem mass spectrometry in time or tandem mass spectrometry in space.

[053] in one embodiment, the methods of the invention are used to diagnose a disease associated with altered levels of ρΕ3-Αβ. In a further embodiment, the methods of the invention are used to prognose a disease associated with altered levels of pE3~A[i In certain embodiments, the methods of the invention are used in the treatment of a disease associated with altered levels of ρΕ3-Αβ, for example, by stratifying disease states, monitoring disease progression, and/or assessing responsiveness to treatment, In addition, the methods of the invention may be used to tailor and direct therapies. In some embodiments, the disease is Alzheimer's disease, in particular in the early stages of the disease.

[054] The method can further comprise assessing the efficacy of a therapeutic or a prophylactic treatment, If the method comprises assessing the efficacy of the therapeutic or the prophylactic treatment, the method may further comprise modifying the therapeutic or the prophylactic treatment in order to improve efficacy. In some embodiments, the methods of the invention are used to determine whether a subject predisposed to or suffering from a given disease will benefit from treatment and further optionally encompasses selecting or identifying candidates for therapy. In some embodiments, the methods of the invention may he used to determine whether a subject having or at risk for a given disease is a candidate for therapy. For example, the subject may have experienced some symptom of the disease, may have been diagnosed as having or being at risk for the disease, and/or demonstrates an unfavorable

concentration or amount of ρΕ3-Αβ or a fragment thereof, as described herein, In some embodiments, the methods of the invention may be used to determine whether one or more subjects exhibit unfavorable concentrations of ρ£3-Αβ, e.g., less than about 0.33 pg/mL; about 0.33 pg/niL to about 0.39 pg/mL; about 0.33 pg/mL to about 0.48 pg/mL: about 0,33 pg/mL to about 0.57 pg/rnL; about 0,33 pg/mL to about 0.69 pg/mL; or about 0.33 pg/rnL to about 0.87 pg/mL, In certain embodiments, the methods may be used to select, identify, and/or enrich patients for a clinical trial for a disease associated with altered levels of ρΕ3-Αβ. In certain embodiments, the disease is Alzheimer's disease,

[055] The methods of the invention may be used to detect and measure Αβ in biological samples. Biological samples may include a fluid, a cell, or a tissue sample. Biological fluids may include cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, or peritoneal fluid. A ceil sample or a tissue sample may include a biopsy, a tissue, a cell suspension, or other specimens and samples, such as clinical samples. Specimens and samples may be obtained, for example, for the diagnosis, prognosis, and/or treatment of a disease.

[056] In some embodiments, the disease to be diagnosed, prognosed, and/or treated as described above is primary amyloidosis. Diseases of primary amyloidosis include, but are not limited to, Alzheimer's disease, sporadic Alzheimer^'s disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mi id cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type. Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), am otropic lateral sclerosis (ALS), and type 2 diabetes.

[057] In some embodiments, the disease to be diagnosed, prognosed, and/or treated is secondary amyloidosis. Diseases of secondary amyloidosis include, but are not limited to tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.

[058] In some embodiments, the biological sample to be analyzed by mass spectrometry is first purified to reduce interfering compounds i the matrix. Sample purification is intended to improve the bioanalytieal performance of the assay by cleaning up the target of interest. In certain embodiments, the target of interest in the sample is APP, Αβ, or ρΕ3-Αβ. In some embodiments, the target of interest in the sample does not undergo purification. In some embodiments, the target of interest in the sample is purified by non-antibody-based sample purification methods, which may include liquid-liquid extraction (LLE), filtration (partial) protein precipitation (PP), and/or solid-phase extraction (SPE). In SPE, the different components of the sample are dissolved or suspended in solution ("mobile phase"). These different components exhibit different affinities for a solid through which the solution is passed ("solid phase"). In some embodiments, as the mobile phase passes through the solid phase, undesired components of the mobile phase may be retained by the solid phase (i.e., the target of interest in the mobile phase is purified). In other embodiments, desired components may be retained by the solid phase (i.e., the target of interest is retained in the solid phase), and a second mobile phase is used to elute the retained target of interest for further processing or analysis, in some embodiments, LLE, PP, and/or SPE is combined with other purification methods. In certain embodiments, the SPE step is followed by size exclusion chromatography or SDS-PAGE.

[059] In some embodiments, the target of interest in the sample is purified by antibody-based sample purification methods. In certain embodiments, the sample is purified by inimunodepletion, which is intended to extract potential interfering peptides and leave the target of interest in a cleaner medium. In certain embodiments, albumin, immunoglobulin, haptoglobin, and/or fibrinogen is immunodep!eted, e.g., by commercially available kits, in some embodiments, the sample is not purified by immunodepletion.

[060] In certain embodiments, the sample is purified by immunocapture, which is intended to enrich the target of interest in the sample, in some embodiments, the immunocapture is combined with purification techniques after an enzymatic digest and prior to liquid chromatography mass spectrometry,

Immiinoprecipitation

[061] In some embodiments, the immunocapture is mmvunopreeipitation. In certain embodiments, at least one pE3~Aj¾~specific antibody is used to capture pE3-Aji. In one aspect of the embodiments, the at least one pE3-Aj3-specifie antibody is covalently or non-covalentiy conjugated to a magnetic bead, a polystyrene bead, or a stationary phase sorbent column, e.g., using a commercially available kit. In certain embodiments, the pES-Ap-specific antibody is conjugated to a magnetic bead to form an antibody-bead immunocomplex. In one aspect of these embodiments, the antibody- bead irnmunocompiex binds to and immunoprecipitates ρΕ3-Αβ in a biological sample. The antibody-bead-pl:'3-A|3 complex is separated magnetically, and ρΕ3-Αβ is eluted. ρΕ3-Αβ-δρεοΐίίο antibodies include, but are not limited to:

ABl OO-1 1

9D5 (DSM ACC3056)

8C4 (DSM ACC3066)

AB 5-5-6 (DSM ACC2923)

AB 6-1 -6 (DSM ACC2924)

AB 17-4-3 (DSM ACC2925)

AB 24-2-3 (DSM ACC2926)

B 121, (SEQ n) NOs: 7 and 8)

R17L (SEQ ID NOs: 7 and 9)

hE8L (SEQ ID NOs: 7 and 10)

CI-C7 {SEQ I D NOs: 1 .1 and 12)

[062] See, e.g., Wirths et al., 2010, WO2011 151076, Frost et al., 2012, WO2012021469, and Mandler et al., each incorporated herein by reference for its disclosure of one or more of these antibodies.

[063] In some embodiments, the immunoprecipitaiion uses an antibody that is specific for ρΕ3-Αβ. In some embodiments, the antibody concentration ranges from about 0.1 ^ig/mL to about 2 [ig/mL; about 2 μ§/ηιΕ to about 5 μg/mL·; or about 5 to about 10 Lig/'mL. The skilled artisan would appreciate that pEl l-Αβ or other ρΕ-Αβ species could be detected by exchanging the pE3~Ap~specific antibody for a pE1 1 -Αβ- specific antibody (see, e.g., Perez-Garmendia et al., 2010; commercially available NOP 1-44070) or other ρΕ-Αβ-specific antibodies. Enzymatic digestion

[064] In some embodiments, the target of interest in the sample is

enzymatically digested. In one aspect of these embodiments, the enzymatic digestion process comprises denaturation, reduction, alkylation, and/or enzymatic cleavage, The enzymatic cleavage is intended to consolidate signal over fewer charge states and isotopic distributions and thus improve the sensitivity of rnass-to-charge ratio measurements.

[065] In some embodiments, the target of interest in the sample is denatured and unfolded into single-stranded structures, which are more accessible to the digestion enzyme. In certain embodiments, the denaturing agent is urea, in some embodiments, the target of interest in the sample is not denatured.

[066] In some embodiments, the target of interest in the sample is reduced to break the disulfide bonds, making the target of interest in the sample more accessible to the digestion enzyme, in certain embodiments, the reducing agent is dithiothreitol (DTT), In some embodiments, the reducing agent is im(2~carboxyethyl)phosphine. In some embodiments, the target of interest in the sample is not reduced.

[067] In certain embodiments wherein the target of interest in the sample is reduced, the peptide is alkylated to prevent the formation of new, non-specific disulfide bonds. In some embodiments, the alkylating agent is iodoacetamide. In some embodiments, the alkyiation step is performed in dark conditions. In some

embodiments, the target of interest in the sample is not alkylated.

|068| In certain embodiments, the target of interest in the sample is cleaved by at least one proteolytic enzyme. In alternate embodiments, the target of interest in the sample is cleaved by microwave-assisted formic acid hydrolysis. Examples of proteolytic enzymes include, but are not limited to, Lys-N, Lys-C, thermolysin, G!u-C, CNBr, Arg-C, Asp-N, proteinase K. elastase, trypsin, chymotrypsin, and pepsin, in a certain embodiment, die target of interest in the sample is digested with Lys-C. in some embodiments, the sample undergoes a first purification prior to enzymatic digestion and/or a second purification after enzymatic digestion in a "double clean-up" procedure, in some embodiments, the sample undergoes a first immunocapture step prior to enzymatic digestion and/or a second immunocapture step after enzymatic digestion.

[069] In some embodiments, the target of interest in the sample is not enzymatically digested, in particular, certain peptides with molecular weights between 4 - 16 k^'Da may not require enzymatic digestion (van den Brock et al., 2013).

Separation by chromatography

| 70] in some embodiments, the target of interest in the sample is separated into components by liquid or gas chrom tography, a process by which a mixture carried by a liquid or a gas is separated as a result of differential distribution of the chemical entities as they flow over a stationary liquid or solid phase. Separation is intended to reduce ionization suppression and mass spectrometry interferences. In some

embodiments, the separation may increase analysis speed and sample throughput.

[073 ] in some specific embodiments, the target of interest in the sample is separated by liquid chromatography (LC). Targets are separated on columns of various sizes with particles of various sizes. In general, smaller particles increase separation efficiency, but impair (increase) back pressure. In some embodiments, the particles are semi-porous particles, which are intended to decrease hack pressure. In some embodiments, the columns are stationary phase sorbent columns, C8 columns, C I 8 columns, fused core columns, or monolithic columns. In certain embodiments, the column is an ACE 5 phenyl, 2.1 x 50 mm column, In some embodiments, the column temperature is above 35°C. In general, increased column temperature improves separation efficiency. Examples of LC include, but are not limited to, normal phase liquid chromatography (NPLC), reverse phase liquid chromatography (RPLi ^"}. high performance liquid chromatography (HPLC), ultra high performance liquid

chromatography (UHPLC), high turbulenc liquid chromatography (HTLC), and multidimensi nal liquid chromatography, in one embodiment, the sample is separated by HPLC. In HPLC, the degree of separation is increased by forcing the mob le phase through a stationary phase (e.g., a densely packed column) under pressure.

Detection by. mass spectrometry and uses thereof

[Θ72] In some embodiments, the methods of the invention are used to determine the concentration or amount of ρΕ3-Αβ in a biological sample that, contains low levels of ρΕ3-Αβ, such as samples that are very dilute and/or samples of very limited volume. In particular, the invention may be used to delect and measure ρΕ3-Αβ in cerebrospinal fluid. In some embodiments, the methods of the invention may be used to determine whether one or more subjecis exhibit unfavorable concentrations of pE3- Αβ, e.g., less than about 0.33 pg mL; about 0.33 pg/mL to about 0.39 pg/mL; about 0.33 pg/mL to about 0.48 pg/mL: about 0.33 pg/raL to abou 0.57 pg/mL; about 0.33 pg/mL to about 0.69 pg/mL; or about 0,33 pg/mL to about 0.87 pg/mL.

[073] In some embodiments, the target of interest in the sample is analyzed by mass spectrometry, in particular tandem mass spectrometry (MS/MS). The sample is ionized to generate analyte ions with net electrical charge. In some embodiments, the sample is ionized by atmospheric pressure chemical ionization (APCI). In some embodiments, the sample is ionized by electrospray ionization, in certain embodiments, the detection instrument is a triple quadrupole (QqQ) instrument. In one embodiment, the instrument operates in selective reaction monitoring (SRM) mode. In another embodiment, the instrument operates in multiple reaction monitoring (MRM) mode. First, precursor ions are filtered by mass-to-charge ratio in a first mass filter (Ql ). Next, analyte ions collide with gases in a collision cell (q2) in a collision-induced dissociation step, which further fragments the analyte ions. In some embodiments, the gas may be argon, xenon, nitrogen, or helium. Third, fragment ions are filtered by mass-to-charge ratio in a second mass filter (Q3). The fragment ions are detected by a detector, and the molecular weight and the mass-to-charge ratio are calculated (FIG. 1). in alternate embodiments, fragment ions are filtered by mass-to-charge ratio and detected as electronic signals, in certain embodiments, the mass spectrometry is tandem mass spectrometry in time or tandem mass spectrometry in space.

[074] In some embodiments, the methods of the invention are used to determine the concentration or amount of ρΕ3-Αβ in a biological sample. The biological sample is subjected to ionization to generate one or more charged ions, and the intensity of the one or more charged ions is measured. In some embodiments, at least three additional samples containing known concentrations of ρΕ3-Αβ (e.g., standards, other biological .samples) are each subjected to ionization to generate one or more charged ions. The intensities of the charged ions arc measured for each sample and used to generate a standard curve comprising the at least three known

concentrations of ρΕ3-Αβ. The concentration of ρΕ3-Αβ in the biological sample is calculated by comparing the intensity of the ions of the biological sample to the at least three known concentrations of the standard curve.

[075] In some embodiments, the methods of the invention are used to compare concentrations of ρΕ3-Αβ in two or more biological samples. Two or more biological samples are analyzed by MS as described above and concentrations of ρΕ3-Αβ are measured. In some embodiments, the two or more biological samples are collected from different human subjects. It is known, for example, that CSF concentrations of unmodified Αβ are lower in AD patients (Mehta et al., 2000: Blennow and Hampel, 2003), possibly due to increased Αβ aggregation the brain (Fagan et al., 2006; Strozyk et al., 2003), but concentrations of ρΕ3-Αβ in the CSF of AD patients were previously unknown. In one embodiment, the methods of the invention are used to detect and measure ρΕ3-Αβ in CSF samples collected fr m AD patients and healthy controls, which are assayed by MS as described above. Analyses of ρΕ3~Αβ in CSF in FIG- 11 demonstrate that the concentration of ρΕ3-Αβ in AD patients is significantly lower than the concentration of ρΕ3-Αβ in control patients. In alternate embodiments, relative levels of ρΕ3-Αβ are determined and compared, in certain embodiments, relative levels of ρΕ3-Αβ are determined without using standards and without generating a standard curve.

[076] In some embodiments, CSF samples may be collected from at least one AD patient, at least one healthy control, and at least one undiagnosed subject. The samples are assayed by MS as described above. The ρΕ3-Αβ concentration of the at least one undi agnosed subject may be compared to the ρΕ3-Αβ concentration of the at least one AD patient and the at least one healthy control. In alternate embodiments, relative levels of ρΕ3-Αβ are measured and compared.

[077] In some embodiments, the two or more biological samples, e.g.. CSF and plasma, are collected from the same human subject, and concentrations of ρΕ3~Αβ are measured and compared. It is known, for example, that low concentrations of unmodified Αβ in the CSF may be indicative of increased Αβ aggregation in the Αβ plaques of the brain (Fagan et al.. 2006; Strozyk et al., 2003). in alternate

embodiments, relative levels of ρΕ3-Αβ are measured.

[078] In some embodiments, the methods of the invention are used to detect and measure ρΕ3-Αβ after an initial screen for ρΕ3~Αβ. In one embodiment, the initial screen is an immunoassay, in particular an immunoPCR. For example, the

immunoassay detects the presence of ρΕ3-Αβ in a biological sample wherein the presence of ρΕ3-Αβ is indicative of a disease, and the ρΕ3-Αβ concentration is measured by the methods described herein. In biological samples wherein the absence of ρΕ3-Αβ is detected by the immunoassay, a second biological sample is collected from a different tissue, and the ρΕ3-Αβ concentration is measured by the methods described herein. In certain embodiments, the methods of the invention are used to detect and measure ρΕ3-Αβ before, at the same time as, or after detecting and/or measuring ρΕ3~Αβ using the immunoassay.

[079] In some embodiments, the methods of the invention may be used to detect, but not to measure ρΕ3-Αβ. For example, the methods are used to determine the presence or absence of ρΕ3-Αβ. In some embodiments, the presence of ρΕ3-Αβ is determined as a level that is at or above the LOD. In some embodiments, the presence of ρΕ3~Αβ is determined as a level that is at or above the LOQ, In some embodiments, the absence of ρΕ3-Αβ Is determined as a level that is below the LOD. In some embodiments, the absence of ρΕ3-Αβ is determined as a level that is below the LOQ. in biological samples wherein the presence of ρΕ3-Αβ is indicative of a disease, the sample is further evaluated by an alternative quantitative method. In biological samples wherein the absence of ρΕ3-Αβ is indicative of a disease, a second biological sample is collected from a different tissue. The second sample is analyzed for the presence or absence of pE-3-Αβ, as described above, and/or by an alternative quantitative method.

[080] The methods of the invention may be used in the diagnosis, prognosis, and treatment of disorders, such as primary or secondary amyloidosis, which may be associated with altered levels of ρΕ3-Αβ. In addition, the methods of the invention may be used to tailor and direct therapies. In some embodiments, the disease is Alzheimer's disease, in particular in the early stages of the disease. In certain embodiments, the methods of the invention are used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment. In some embodiments, the methods of the invention may be used to determine whether a subject having or at risk for a given disease is a candidate for treatment. For example, the subject may demonstrate an unfavorable concentration or amount of ρΕ3-Αβ or a fragment thereof, as described herein. In certain embodiments, the methods may be used to select, identity, and/or enrich patients for a clinical trial for a disease associated with altered levels of ρΕ3-Αβ. in certain embodiments, the disease is Alzheimer's disease.

Kits

[081 ] The reagents of the invention can be provided in a kit (i.e., a packaged combination of reagents with instructions for performing the assay) for use in conjunction with a mass spectrometer. The kit may include antibodies, antibody complexes, substrates, and/or cofactors. In addition, additives such as stabilizers, buffers, and other solutions may be included. The relative amounts of the reagents may be varied to provide for concentrations which optimize die sensitivity of the assay.

[082] In a specific embodiment, the kit is a diagnostic kit. The diagnostic kit may be used for the detection and diagnosis of ρΕ3-Αβ diseases and conditions, particularly Alzheimer's disease.

i..;Lsi. of Exemplary _. Embo_djments

1 . A method of detecting ρΕ3-Αβ in a biological sample comprising

(a) subjecting the biological sample to ionization to generate one or more charged ions;

(b) detecting the presence or absence of the one or more charged ions of (a) by mass spectrometry; and (c) using ihe deiecied ions of (b) to determine the presence or absence of pE3- Αβ in the biological sample. ethod of measuring ρΕ3-Αβ in a biological sample comprising

(b) measuring the intensity of the one or more charged ions of (a) by mass spectrometry; and

(c) using the intensity of (b) to determine the amount of ρΕ3-Αβ in the biological sample. method of embodiment 2, further comprising

(a) .subjecting at least three additional samples containing known concentrations of pE^'3-Αβ to ionization to generate three or more charged ions and measuring the intensities of the charged ions by mass spectrometry;

(b) using the intensities of (a) to generate a standard curve comprising the at least three known concentrations of ρΕ3-Αβ; and

(c) calculating the concentration of ρΕ3-Αβ in the biological sample by comparing the intensity of the charged ions of the biological sample to the standard curve. method of embodiment 2, further comprising

(a) subjecting at least one additional biological sample to ionization to generate one or more charged ions and measuring the intensity of the one or more charged ions by mass spectrometry; (b) comparing the relative intensities of the charged ions for each of the two or more biological samples; and

(c) determining the relative amounts of pE3-Ap in the two or more biological samples.

5. The method of any one of embodiments 1 -4, wherein the ionization is eleeirospray ionization or atmospheric pressure ionization,

6. The method of any one of embodiments 1 -5, wherein the ionization generates at least one charged ion, further wherein at least one of the charged ions consists of a charge of 2- , 3+, 4-h or 5+.

7. The meihod of any one of embodiments 1 -6, wherein the ionization generates at least one charged ion, further wherein at least one of the charged ions has a mass-to-charge (m/z) ratio between 10 and 2000.

8. The meihod of any one of embodiments 1-4, wherein the mass spectrometry is tandem mass spectrometry (MS/MS).

9. The method of embodiment 8, wherein the mass spectrometry uses multiple reaction monitoring (MRM) or selective reaction monitoring (SRM).

10. The method of any one of embodiments 1 -4, further comprising purification of the biological sample in preparation for mass spectrometry, wherein the purification is selected from the group consisting of (a) solid-phase extraction (SPE):

(b) restriction enzyme digestion;

(c) immunoprecipitaiion;

(d) chromatography; and

(e) chemical hydrolysis,

1 1. The method of embodiment 10, wherein the restriction enzyme digestion comprises at least one restriction enzyme that cleaves ρΕ3-Αβ into at least one fragment between 3 and 25 amino acids in length.

12. The method of embodiment 10 or 1 1 , wherein the restriction enzyme digestion comprises at least one restriction enzyme selected from the group consisting of Lys-N, Lys-C, thermolysin, Glu-C, CNBr, Arg-C, Asp-N, proteinase K, elastase, trypsin, chymoirypsin, and pepsin.

13. The method of any one of embodiments 10-12, wherein the restriction enzyme digestion comprises the restriction enzyme Lys-C.

14. ^'i^'he method of embodiment 10, wherein the immunopreeipitation comprises at least one pE3-Ap-speeifie antibody selected from the group consisting of A Bl 00-1 1 , 9D5, 8C4, AB 5-5-6, AB 6-1 -6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hESL, and R17L.

15. The method of embodiment 14, wherein the pE3-Ap-speeific antibody is AB 100-1 1 and/or 9D5. 16. The method of embodiment 14 or 1 5, wherein the the concentration of the ρ£3-Αβ- specific antibody is 0.3 - 1 0 ;ig/mL_v

17. The method of any one of embodiments 14- 16, wherem the pE3-A3-specifie antibody is covalentiy or non-cova!ently conjugated to a magnetic bead, a polystyrene bead, or a stationary phase sorbeni column.

18. The method of embodiment 10, wherein the chromatography is liquid

chromatography or gas chromatography.

19. The method of embodiment 1 8, wherein the liquid chromatography is selected from the group consisting of normal phase liquid chromatography (NPLC), reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (IJHPLC), high turbulence liquid

chromatography (HTLC), and multidimensional liquid chromatography.

20. The method of embodiment 18 or 19, wherein the liquid chromatography is HPLC

21 . The method of any one of embodiments 1 -4, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, peritoneal fluid, a biopsy, a tissue sample, a clinical specimen, a eel) sample, and a cell suspension.

22. The method of any one of embodiments 1 -4, wherein the biological sample contains low concentrations of pE3-Ap\ 23. The method of embodhnent 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.87 pg/mL.

24. The method of embodiment 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.69 pg/mL.

25. The method of embodiment 21 or 22, wherein the concentration of pE3-A|3 in the biological sample is 0.33 - 0.57 pg/mL,

26. The method of embodiment 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.48 pg/mL,

27. The method of embodiment 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.39 pg/mL.

28. The method of any one of embodiments 21 -27, wherein the biological sample is cerebrospinal fluid.

29. The method of any one of embodiments 21 -27, wherein the biological sample is plasma,.

30. A method of diagnosing a disease associated with altered levels of ρΕ3-Αβ comprising (a) subjecting a biological sample to ionization to generate one or more charged ions:

(b) detecting the presence or absence of the one or more charged ions of (a) by mass spectrometry; and

(c) using the detected ions of (b) to determine the presence or absence of pE3~ Αβ in the biological sample.

31. A method of prognosing a disease associated with altered levels of ρΕ3-Αβ comprising

(a) subjecting a biological sample to ionization to generate one or more charged ions;

(c) using the detected ions of (b) to determine the presence or absence of pE3- Αβ in the biological sample.

32. A method of developing, tailoring, and/or directing therapies for a disease associated with altered levels of ρΕ3-Αβ comprising

(c) using the detected ions of (b) to determine the presence or absence of pE3- Αβ in the biological sample. 33. The method of any one of embodiments 30-32, further comprising

(a) measuring the intensity of the detected ions; and

(b) using the intensity of (a) to measure the amount or concentration of ρ^'Ε3-Αβ in. the biological sample.

34. T he method of any one of embodiments 30-33, wherein the disease is selected from the group consisting of Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), rniid cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson- Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creuizfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.

35. The method of any one of embodiments 30-34 to determine whether a subject exhibits unfavorable concentrations of ρΕ3-Αβ associated with a disease, wherein the concentration of ρΕ3-Αβ is about. 0.33 pg/mL to about 0,87 pg/roL; about 0.33 pg/m L to about 0,69 pg mL; 0.33 pg/mL to about 0.57 pg/mL: 0.33 pg/mL to about 0.48 pg/mL; or about 0.33 pg/mL to about 0,39 pg/mL,

36. A kit for assaying a biological sample for the presence, absence, or concentration of ρΕ3-Αβ by mass spectrometry, the kit comprising (a) instructions for assaying the biological sample for ρΕ3·-Αβ by mass spectrometry; and

(b) at least one pE3-Af3~specifie antibody of embodiment 14,

37. A method of detecting ρΕ3-Αβ in a cerebrospinal fluid sample comprising

(a) purifying the cerebrospinal fluid sample, comprising

(i) Lys-C restriction enzyme digestion,

(ii) solid-phase extraction,

(iii) immunoprecipitation, and/or

(i v) ebro.m atography ;

(b) subjecting the purified cerebrospinal fluid sample to ionization to generate one or more charged ions:

(e) detecting the presence or absence of the one or more charged ions by raass spectrometry; and

(d) using the detected ions of (c) to determine the presence or absence of pE3~ Αβ in the biological sample,

38. The method of embodiment 37. further comprising

(a) measuring the intensity of the detected ions; and

(b) using the intensity of (a) to measure the amount or concentration of ρΕ3-Αβ in the cerebrospinal fluid sample,

39. The method of embodiment 37 or 38, wherein the immunoprecipitation comprises at least one pE3-AP-specitlc antibody selected from the group consisting of AB 100-1 1. 9D5, 8C4, AB 5-5-6, AB 6-1 -6, AB 17-4-3, AB 24-2-3, B 12L, CI-C7, hE8L, and R i 7L. A method of detecting ρΕ3-Αβ in a cerebrospinal fluid sample comprising

(a) purifying the cerebrospinal fluid sample, comprising

(i) L-ys-C restriction enzyme digestion,

(ii) solid-phase extraction,

(Hi) immunoprecipitation, wherein the ρΕ3-Αβ is imrnunoprecipitated using a pE3-Ap>--specifie antibody, further wherein the pE3-Ap-speeific antibody is AB 100-1 1 , and/or

(iv) chromatography, wherein the chromatography is H PLC:

(c) detecting the presence or absence of the one or more charged ions by tandem mass spectrometry; and

(d) using the detected ions of (c) to determine the presence or absence of pE3~ Αβ in the biological sample.

41. The method of embodiment 40, further comprising

(a) measuring the intensity of the detected ions; and

(b) using the intensity of (a) to measure the amount or concentration of ρΕ3-Αβ in the cerebrospinal fluid sample.

[083] All references cited in this disclosure are incorporated herein in their entirety. Full citations for the scientific literature referenced throughout the specification include: Acero ei al. "immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits." J Neuroimmunol 213(1 -2):39-46 (2009).

Bib! ei al. "Characterization of cerebrospinal fluid aminoterminally truncated and oxidized amyloid-β peptides." Proteomics Clin Appl. 6(3-4): 163-9 (2012).

Biancas-Mejia et al, "Systemic arn. loidoses." Annu Rev Biochem. 82:745-74 (2013).

Blennow et al. "Cerebrospinal fluid bioniarkers for Alzheimer's disease." J Alzheimer s Dis. 1 8(2):413-7 (2009).

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Blennow et al. "CSF markers for incipient Alzheimer's disease." Lancet Neurol.

2(10):605-13 (2003).

De .impe et al. "Intracellular accumulation of aggregated pyroglutamate amyloid beta: convergence of aging and Αβ pathology at the lysosome." Age 35(3):673-87 (2013).

DeMaiios ei al. "In vestigaiion of the cenirai mechanism of action required for antibody- mediated phagocytosis of pre-exisitng plaque and the development of a novel antibody therapeutic for Alzheimer's disease." Alzheimer 's & Dementia 7(4):e55 (201 1), Fagan et al M. ' nverse relation between in vivo amyloid imaging load and

cerebrospinal fluid Abeta42 in humans." Ami Neurol, 59(3): 5 12-9 (2006),.

Frost ei al. "Pyroglutamate-3 amyloid-β deposition in the brains of humans, non-human primates, canines, and Alzheimer disease-like transgenic mouse models." Am J Pathol 183(2):369~81 (2013).

Frost et al . "Passive immunization against pyroglutamate-3 amyloid-β reduces plaque burden in Alzheimer-like transgenic mice: a pilot study.^"' Neurodegener Dis. 10(1 - 4):205-70 (2Q] 2)..

Gienner et al. "Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein." Biochem Biophys Res Commun, 120(3):885-90 (1984).

Gorevic et al. "isolation and partial characterization of neurofibrillary tangles and amyloid plaque core in Alzheimer's disease: imrnunohistological studies." J

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Haass et al . '"Soluble protein oligomers in neurodegeneration : lessons from the

Alzheimer's amyloid beta-peptide." Nat Rev Mo! Celt Biol 8(2): 101 - 12 (2007). ϊ-!arigaya et ai. ''Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer^'s disease brain." Biochem Biophys Res Commun. 276(2):422-7 (2000).

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syndrome." Proc Natl Acad Sci USA. 82(12):4245-9 (1985). Mehta et al. "Plasma and cerebrospinal fluid levels of amyloid beta proteins 1 -40 and 1 - 42 in Alzheimer disease." Arch Neurol. 57(1 ): 100-5 (2000).

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Amyioid-β Load and Cognitive Decline in Alzheimer's Disease." J Alzheimers Dis. (201 3).

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120(2): 185~93 (2010).

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Russo et al. "Ammo-terminal modification and tyrosine phosphorylation of carhoxy- terminal fragments of the amyloid precursor protein in Alzheimer's disease and Down's syndrome brain." Neurobiol Dis. 8(1): 173-80 (2001 ). Saido et al, "Amino- and carboxyl-terminal heterogeneity of beta-amyloid peptides deposited in human brain." Neurosci Lett. 21 5(3): 173-6 ( 1996).

Schilling et al. "On the seeding and oligomerization of pGiu-amyloid peptides (in viiro)." Biochemistry. 45(4.1): 12393-9 (2006),

Schilling et al. "Glutammyl cyclases unfold glutamyl cyclase activity under mild acid conditions/' FEBS Lett. 563(l-3): 191-6 (2004).

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Strozyk et al. "CSF Abeta 42 levels correlate with amyloid-neuropathology in a population-based autopsy study." Neurology. 60(4):652-6 (2003). van den Broek et al. "Bioanalyticai LC-MS/MS of protein-based biopharmaeeutieals." J Chromatogr B Analyt Techno! Biomed Life Sci. 929: 161 -79 (2013).

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[084] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations thai may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

EXAMPLES

[085] The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.). but some experimental errors and deviations should be expeeted.

Example 1 : Internal st ndard, control, and biological samples

[086] Αβι-16, Αβ₃.|₆, ΑβρΒ-30_» and Αβργ-3. 0 were acquired from Anaspec Inc. (Fremont, CA); Αβ ¾8, β|.₄ο, Αβ _?., Αβ^ο, and Αβ_Ρο-42 were acquired from American Peptide Company (Sunnyvale, CA); and 13C6, 15Ν4-Κ5-Αβ_ΡΕ3-ιο was purchased from New England Peptide inc. (Gardner, MA) to serve as internal standards. Lyophilized peptides were reconstituted in dimethyl sulfoxide (DMSO) to concentrations between 0.1 rng/mL and 1 mg/mL and stored at -70^CC until use.

[087] Artificial CSF was purchased from Harvard Apparatus (Holliston, MA), and rat K2EDTA plasma was purchased from Bioreclamation LLC" (Hicksville, NY). The blank matrix was made by inixing artificial CSF and rat plasma at a ratio of 1 ,000:3.

[088] Human cerebrospinal fluid samples from healthy donors and Alzheimer's disease patients were purchased from. Biochemed Services (Winchester, VA) and Bioreclamation LLC (Hicksville, NY).

Example 2: Magnetic beads

[089] A 30 mg/niL magnetic bead suspension (Dynabeads® M-280

Tosylactivated) was vortexed, and 335 μί., of the suspension was immediately transferred to a 1.5 mi, Eppendorf tube. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. 1 mL of 0.1 M

sodiirmphosphate buffer, pH 7.4 (''Buffer B") was added to the tube and vortexed. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. 1 mL of 0.1 M sodiumpbosphate buffer, pH 7.4 ("Buffer B") was added to the tube and vortexed. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. The magnetic beads were resuspended in 100 pL of Buffer B. 84 μΐ, of 2.4 rng/mL monoclonal antibody AB 100-1 1 and 100 pL of 3M ammonium sulfate in Buffer B ("Buffer C) were added, then shaken at 1 ,200 rpm and 37°C for 12-18 hours, The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. 1 rnL of PBS pH 7.4 with 0.1% (w/v) BSA ("Buffer E") was added to the tube and vortexed. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. 1 niL of PBS pH 7.4 with 0.1% (w/v) BSA ("Buffer E") was added to the tube and vortexed. The tube was placed on a DynaMag™~2 magnet for 1 minute, and the supernatant was removed. 1 ml, of Buffer E was added to make a 10 rng/mL stock suspension and stored at 2-8°C until use.

[090] On the day of analysis, the stock suspension was vortexed and pipetted to a 1.5 rnL Eppendorf tube. 1 mL of 0.5% BSA in IX PBS Tween solution was added to block the beads, and the tube was placed on a nutator to shake for 30 minutes. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. 1 mL of IX PBS Tween was added to the tube, and the beads were washed by vortexing. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. The wash was repeated once using 1 mL of I PBS Tween and twice using 1 ml, I X PBS.

[091] The tube was centrifuged at 4,000 rpm or manually turned on the magnet if the beads did not precipitate robustly. The blocked and washed beads were resuspended in IX PBS to make a 10 mg/'mL suspension.

Example 3: Sample preparation

[092] Spiking solutions for ρΕ3-Αβ were freshly diluted from stock solution of β_ΡΕ3- 0 using 1 % ammonium hydroxide (v/^'v). Internal standard spiking solutions for ρΕ3··Αβ were freshly diluted from stock solution of 13C6, I 5N4-R5- using 1 % ammonium hydroxide (v/v). Each calibration standard or QC for ρΕ3-Αβ was prepared by adding 15 pL of spiking solution of ΑβρΓ.3- 0 and 15 pL internal standard spiking solution of heavy ΑβρΕΜο· For each blank or unknown, 15 pL of 1 % ammonium hydroxide or 15 pL of internal standard spiking solution were added to 450 p.L of blank matrix or unknown human CSF sample, respectively. For each double blank, 30 pL of 1% ammonium hydroxide was added to 450 pL of blank matrix. All samples were mixed by vortexing and. incubated at 2~8°C for 30 minutes.

Example 4: Sample extraction

[093] To each double blank, blank, standard, or unknown sample, 300 pi. of lOX digestion buffer and 45 pL of 50 pg/rnL mass spectrometry grade Lysyi

Endopeptidase® (Wako) in 1 X digestion buffer was added. T he sample was then incubated at 22~26°C for 1 -16 hours and quenched. 102 pL of 250 mM citric acid was added to each unknown sample, and 93 pL of 250 mM citric acid was added to each double blank, blank, and standard to adjust the pH to 5.0-6.5. 6 pL of 10 rng/rnL blocked magnetic beads were added to each sample, and the tube was placed on a nutator and shaken at 22-26°C for 2 hours. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was removed. After sequential washing with 1 ml, of IX PBS Tween, 1 mL of IX PBS. 0.4 mL of I PBS, and 0,3 m l of water, the beads were resuspended in 100 pL of 5% acetoniirile and 0.5% formic acid, then incubated at 90°C for 2 minutes. After cooling down, the tube was centrifuged at 4.000 rpra for 1 minute. The tube was placed on a DynaMag™-2 magnet for 1 minute, and the supernatant was transferred into a 96- well plate for injection.

Example 5: Li uid chromatography and mass spectrometry

[094] The HPLC system was a Waters Acquity i-class U PLC consisting of a sample manager-FTN and a binary solvent manager. An ACE 5 phenyl, 2.1 x 50 mm column was used at 30°C. Mobile Phase A was 0.1 % acetic acid in water: mobile Phase B was 0.1 % acetic acid in acetonitrile. The LC method is detailed in Table 1.

Table 1 : LC method for pE3 amyloid beta assay

[095] The detector was a Waters Xevo TQ-S triple quadrupole mass

spectrometry running R.M in positive ESI mode. The resolution for both Ql and Q3 was set to 1.5. The MRM transitions are listed in Table 2 and MS parameters in Table

Ta tble 2: MR M transitions for pE3 m y!oid beta assay

! Heavy pE3~ 16 ! 441 ,3500 -> 557.5500 1 14 I 0.05 I

Table 3: MS parame ers for pE3 amyloid beta assay

Capillary Voiiage:: 3.00 kV

Source Offset Voltage: 50 V

Desolvation Temperature; 650°C

Desoivati n Gas: 800 L/Hr

Cone Gas: 1 50 L/hr

Nebuliser:: 7.0 Bar

Collision Gas: 0.23 mL/mirt

[096| Data were considered acceptable if the following criteria were met: i) at least. 75% of the total number of calibration standards were within ±15% (±20% at LLOQ) of their nominal concentrations, and 2) at least 66% of the total number of quality control samples and at least 50% of QCs per levei were within ±15% of their nominal concentrations.

Example 6: Mass spectrometry analysis

[097] Results were calculated using peak area ratios. Calibration curves were generated using a weighted (l/x2) linear least-squares regression. Calibration standards were analyzed at the beginning and end of each of the analytical sequences. The results for the QC samples were evaluated and indicate that the method performed adequately for this study. The software application used to acquire and process the data for this study was MassLynx V4.1 (Waters Inc. Milford, MA).

Example 7: LC-MS/MS analysis with solid-phase extraction

[098] Biological samples were pre-treated prior to solid-phase extraction (SPE). 10 pg/raL of Αβ _Κ. , A|3_p -4O, Αβ _¾ Αβ ο, Αβ|.₃₈, and Αβ_ρ|^₃₀ were diluted in a surrogate matrix comprising 0.3% rat plasma in artificial CSF. Internal standards were added, and the samples were incubated on ice for 30 minutes. 800 μΐ., of 9.5 urea was added and shaken at 50° C for 30 minutes. 20 LIL of 85% H3PO4 was added.

[099] A Waters Oasis® MCX pEIution plate was placed on a vacuum manifold, conditioned using 300 pL of MeOlL and equilibrated using 300 p L of 0.5% H₃PO₄, 800 pL of the pre -treated biological samples, described above, were added to the plate. The plate was washed with 600 μϊ, of 0.5% H3PO4; 400 μΐ, of 0.4% H3PO4 and 40% acetonitrile; 400 pL of 0.4% H₃PO₄ and 80% acetonitrile; and 600 ί· of water.

[0100] The sample was eluied using two washes of 30 p.L of 45% acetonitrile and 1 % NH4OH. The eiuant was diluted in 40 μΐ, of water. FIG. 4 shows standard curves of Αβ|.₃₈, Αβ_ϊ- ο, Αβ]„ι₂, Αβ_ΡΕ5.₃ο, Αβ_ρΒ-40₅ and Αβ_ρ£3- 2 using solid-phase extraction. A calibration curve of 10 pg/mL - 8 ng/mL is achieved.

[0101] Unmodified Αβ was detected in human CSF (FIGS, 5A-C), Αβ|.₄₂ was present at 0.47 ng/mL. Αβ] . β was present at 5.7 ng/mL. Αβ|.₃8 was present at 2.2 ng/mL. FIGS. 5A-C show particular Αβ species that were detected in human CSF. Αβι.42, Αβι-4 , Αβι-38 were detected in pooled human CSF without using solid-phase extraction (FIG. 5A). ΑβρΓ_;3-30 and Αβ_ΡΕ3-42 were below the limit of detection (LOD), and Αβ_ΡΕ34ο was detected but below the limit of quantitation (LOQ) in pooled human CSF after using solid-phase extraction (FIG. SB).

Exam le 8: LC-MS/MS analysis with Lys-C digestion and solid-phase extraction

[01021 Biological samples were digested using a restriction enzyme prior to solid-phase extraction (SPE). 200 pL of a biological sample was incubated with 200 μί., of 50 m Tris, 2 mM EDTA, pH 8.5, and 20 _M L of 100 μ , Wake lysyl

endopeptidase (Lys-C) for 1 -16 hours at 22-26°C. 5 pL 85% H3PO4 was added. Lys-C cleaves at the C-terminus of a lysine residue (K), which is located at position 1 6 of Αβ (FIG. 6),

[0103] A Waters Oasis® MCX pElution plate was placed on a vacuum manifold, conditioned using 300 p.L of MeOH, and equilibrated using 300 uL of 0.5% H3PO4. 200 μΣ, of the digested samples, described above, were added to the plate. The plate was washed with 600 μΐ, of 0.5% H₃P0₄; 300 uL of 0.4% H3PO4 and 40% acetomtrile; 300 μΐ, of 0,4% i i₃P(>₄ and 80% acetonitrile; and 600 pL of water.

[0104] The sample was eluted using two washes of 30 μϊ_. of 8% acetonitrile and 10% NH4OH. The eluant was diluted in 30 μΐ, of water and analyzed by LC-MS/MS. FIG. 7 shows chromatograrns of 20 pg/mL (45 pL) of Αβ_ΡΕ3-ΐ6> 20 pg/mL (45 μΐ,) of Α |.|6, 2 pg/mL (45 pL) of Αβ_ρκ_3-] ₆, and 2 pg ^'mL (45 μΐ.) of Αβι..ΐ 6 in pure solvent. Exam le 9: LC-MS/MS analysis with Lys~C digestion, solid-phase extraction, mid isHMMO reci itaiioa

[0105] Biological samples containing 200 pg/mL of Αβ_ΡΕ3-40 were digested using the restriction enzyme Lys-C prior to solid-phase extraction, as described above. β_ΡΕ3-; in the sample was captured by magnetic Dynabeads® conjugated to ABl 00-1 1 . as described above. The sample was washed, and Ap_pE3.,':₆-beacl complex was isolated. The Αβ_ΡΕ3-!ό was released from the Dynaheads® by heat, acidification, and organic solvent washing.

[0106] CSF containing 1 50 μΐ... of 4 pg/mL - 2 ng/mL of of Αβ_ρΕ3-ι ₆ ( FIG. 8), 2 pg/mL - 20 pg/mL Αβ_ΡΕ3-ΐ 6 in surrogate matrix/pooled CSF and individual CSF samples (Table 4), or 800 pL of 2.4 pg/mL - 9.6 pg/mL of Αβ_Ρ .4₀ (FIG. 9) was purified and analyzed by LC-MS/MS using the methods described above. Table 4: Optimized Lys-C digestion and isimiisnoprecspitaiion in CSF samples

[0107] The methods of the invention may be used to detect and measure ρΕ3-Αβ in cerebrospinal fluid, which is challenging using current methods, ρΕ3-Αβ in CSF samples was detected and measured using Lys-C digestion, SPIT immunoprecipitation, and mass spectrometry, as described above. Αβ_ΡΕ3- 0 was detected in individual CSF samples (Table 5). The limit of detection (LOD) for Αβ_ΡΕ3- ο was 0.33 pg mL and the limit of quantitation (LOQ) for Αβ_Ρ - 0 was 0.67 pg mL (Table 5). The sensitivity and specificity of the assay are shown in Table 6 and FIG. 10. Table 5; Optimized Lys-C digestion and immunoprecipitation in CSF sam les

Table 6: Sensitivity and specificity of the biomarker ρΕ3-Αβ

[0108] Analysis of pEJ-Αβ in CSF denionsirates thai the concentration of pE3- Αβ in AD patients is significantly lower than the concentration of ρΕ3-Αβ in control patients (FIG. 1 1 ).

Claims

We claim:

1 , A method of detecting ρΕ3-Αβ in a biological sample comprising

(c) using the detected ions of (b) to determine the presence or absence of the ρΕ3-Αβ in the biological sample.

2, A method of measuring ρΕ3-Αβ in a biological sample comprising

(c) using the intensity of (b) to determine the amount of ρΕ3~Αβ in the biological sample.

3, The method of claim 2, further comprising

(a) subjecting at least three additional samples containing known concentrations of ρΕ3-Αβ to ionization to generate three or more charged ions and measuring the intensities of the charged ions by mass spectrometry;

(b) using the intensities of (a) to generate a standard curve comprising the at least three known concentrations of ρΕ3-Αβ; and (c) calculating the concentration of ρΕ3~Αβ in the biological sample by comparing the intensity of the charged ions of the biological sample to the standard curve.

4. The method of claim 2, further comprising

(a) subjecting at least one additional biological sample to ionization to generate one or more charged ions and measuring the intensi ty of the one or more charged ions by mass spectrometry;

(b) comparing the relative intensities of the charged ions for each of the two or more biological samples; and

(c) determining the relative amounts of ρΕ3~Αβ in the two or more biological samples.

5. The method of any one of claims 1-4, wherein the ionization is eleetrospray ionization or atmospheric pressure ionization.

6. The method of any one of claims 1-5, wherein the ionization generates at least one charged ion, further wherein at leas t one of the charged ions consists of a charge of 2+, 3+, 4+, or 5+.

7. The method of any one of claims 1-6, wherein the ionization generates at least one charged ion, further wherein at least one of the charged ions has a mass-to-charge (m/z) ratio between 10 and 2000.

8. The method, of any one of claims 1-4, wherein the mass spectrometry is tandem mass spectrometry (MS/MS).

9. The method of claim 8, wherein the mass spectrometry uses multiple reaction monitoring (MRM) or selective reaction monitoring (SRM).

10. The method of any one of claims 1-4, further comprising purification of the biological sample in preparation for mass spectrometry, wherein the purification is selected from the group consisting of

(a) solid-phase extraction (SPE):

(b) restriction enzyme digestion;

(c) immunoprecipitation;

(d) chromatography; and

(e) chemical hydrolysis,

11. The method of claim 10, wherein the restriction enzyme digestion comprises at least one restriction enzyme that cleaves ρΕ3-Αβ into at least one fragment between 3 and 25 amino acids in length,

12. The method of claim 10 or 11, wherein the restriction enzyme digestion comprises at least one restriction enzyme selected from the group consisting of Lys-N, Lys-C, thermolysin, Glu-C, CNBr, Arg-C, Asp~N, proteinase K, elastase, trypsin,

chymotrypsin, and pepsin.

13. The method of any one of claims 10-12, wherein the restriction enzyme digestion comprises the restriction enzyme Lys~C,

14. The method of claim 10, wherein the immunoprecipitation comprises at least one pE3-AP~specific antibody selected from the group consisting of ABlOO-l 1, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.

15. The method of claim 14, wherein the pE3-AP-specific antibody is AB 100-11 and/or 9D5,

16. The method of claim 14 or 15, wherein the the concentration of the ρΕ3-Αβ- speeific antibody is 0,1 - 10 μg/ nL,

17. The method of any one of claims 14-16, wherein the pE3-Ap~specific antibody is covalently or non-covalently conjugated to a magnetic bead, a polystyrene bead, or a stationary phase sorbent column.

18. The method of claim 10, wherein the chromatography is liquid chromatography or gas chromatography,

19. The method of claim 18, wherein the liquid chromatography is selected from the group consisting of normal phase liquid chromatography (NPLC), reverse phase liquid chromatography (RPLC), high performance liquid chromatography ( I f PLC), ultra high performance liquid chromatography (IJHPLC), high turbulence liquid cliiOmatography (HTLC), and multidimensional liquid chromatography.

20. The method of claim 18 or 19, wherein the liquid chromatography is HPLC,

21. The method of any one of claims 1-4, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, peritoneal fluid, a biopsy, a tissue sample, a clinical specimen, a cell sample, and a cell suspension.

22. The method of any one of claims 1-4, wherein the biological sample contains low concentrations of ρΕ3-Αβ.

23. The method of claim 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.87 pg/mL.

24. The method of claim 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0,33 - 0.69 pg/mL.

25. The method of claim 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.57 pg/mL.

26. The method of claim 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.48 pg/mL.

27. The method of claim 21 or 22, wherein the concentration of ρΕ3-Αβ in the biological sample is 0.33 - 0.39 pg/mL.

28. The method of any one of claims 21-27, wherein the biological sample is cerebrospinal fluid,

29. The method of any one of claims 21-27, wherein the biological sample is plasma,

30. A method of diagnosing a disease associated with altered levels of ρΕ3~Αβ comprising

(c) using the detected ions of (b) to determine the presence or absence of pE3- Αβ in the biological sample,

33. The method of any one of claims 30-32, further comprising

(a) measuring the intensity of the detected ions; and

(b) using the intensity of (a) to measure the amount or concentration of ρΕ3~Αβ in the biological sample.

34. The method of any one of claims 30-33, wherein the disease is selected from the group consisting of Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson- Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever,

35. The method of any one of claims 30-34 to determine whether a subject exhibits unfavorable concentrations of ρΕ3-Αβ associated wit a disease, wherein the concentration of ρΕ3-Αβ is about 0.33 pg/rnL to about 0.87 pg/mL; about 0,33 pg mL to about 0,69 pg/mL; 0,33 pg/mL to about 0,57 pg/mL; 0.33 pg/mL to about 0.48 pg/mL; or about 0.33 pg/mL to about 0.39 pg/mL.

36. A kit for assaying a biological sample for the presence, absence, or concentration of ρΕ3-Αβ by mass spectrometry, the kit comprising

(a) instructions for assaying the biological sample for ρΕ3~Αβ by mass spectrometry; and

(b) at least one Ε3-Αβ-5 ε€.ΐίΙο antibody of claim 14.

37. A method of detecting ρΕ3-Αβ in a cerebrospinal fluid sample comprising

(a) purifying the cerebrospinal fluid sample, comprising

(i) Lys-C restriction enzyme digestion,

(ii) solid-phase extraction,

(iii) immunoprecipitation, and/or

(iv) chromatography;

(b) subjecting the purified cerebrospinal fluid sample to ionization to generate one or more charged ions;

(c) detecting the presence or absence of the one or more charged ions by mass spectrometry; and

(d) using the detected ions of (c) to measure the presence or absence of ρΕ3-Αβ in the biological sample.

38. The method of claim 37, further comprising

(a) measuring the intensity of the detected ions; and

(b) using the intensity of (a) to determine the amount or concentration of pE3- Αβ in the cerebrospinal fluid sample,

39. The method of claim 37 or 38, wherein the immunoprecipitation comprises at least one pE3-A -specific antibody selected from the group consisting of AB 100-1 1 , 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.