WO2015191825A1 - Méthodes pour la détection et la mesure de la protéine beta amyloïde dans des échantillons biologiques - Google Patents

Méthodes pour la détection et la mesure de la protéine beta amyloïde dans des échantillons biologiques Download PDF

Info

Publication number
WO2015191825A1
WO2015191825A1 PCT/US2015/035282 US2015035282W WO2015191825A1 WO 2015191825 A1 WO2015191825 A1 WO 2015191825A1 US 2015035282 W US2015035282 W US 2015035282W WO 2015191825 A1 WO2015191825 A1 WO 2015191825A1
Authority
WO
WIPO (PCT)
Prior art keywords
biological sample
mass spectrometry
disease
charged ions
sample
Prior art date
Application number
PCT/US2015/035282
Other languages
English (en)
Inventor
Chaoran Ron HUANG
Tao Ye
Liyu Yang
Original Assignee
Biogen Ma Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biogen Ma Inc. filed Critical Biogen Ma Inc.
Publication of WO2015191825A1 publication Critical patent/WO2015191825A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the disclosure relates to methods for detecting and measuring amyloid ⁇ in biological samples and diagnostic, prognostic, and therapeutic uses thereof.
  • the disclosure relates to highly sensitive multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/ S) methods for detecting N- terminal truncated amyloid ⁇ , e.g., pyrogiutamate amyloid ⁇ .
  • LC-MS/ S highly sensitive multiplexed liquid chromatography-tandem mass spectrometry
  • 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).
  • Amyloid aggregates can become insoluble, toxic, and important to disease progression (Blancas- Mejia and Ramirez-Alvarado, 201 3; Hazenberg 2013: WO201 1076854: US8512677).
  • AD amyloid ⁇
  • ⁇ amyloid ⁇
  • AD is a neurodegenerative disease characterized by ⁇ 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.
  • ⁇ plaques i the AD brain comprise ⁇ oligomers (Haass and Selkoe. 2007), and understanding ⁇ accumulation is believed to be critical for the development of early diagnostic tests and eventual therapeutic agents for AD.
  • ⁇ is derived from the proteolytic cleavage of amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • ⁇ 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,
  • ⁇ 3- ⁇ is recognized in the art as one of the most toxic and aggregation-prone forms of ⁇ (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)
  • ⁇ 3- ⁇ is formed by a multi-step process that begins with the removal of the first two amino acids of ⁇ 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).
  • ⁇ 3- ⁇ exhibits increased
  • ⁇ 3- ⁇ exhibits "up to 250-fold accelerated initial formation of aggregates compared to unmodified ⁇ " and 'is crucial for the initiation of disease' " (Schilling et aL, 2006). Not only does ⁇ 3- ⁇ precede the deposition of unmodified ⁇ in the brain, ⁇ 3- ⁇ exceeds the deposition of unmodified ⁇ and "may account for more than 50% of Ap accumulated in plaques" (Russo et al consult, 2002).
  • ⁇ 3- ⁇ 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 ⁇ 3- ⁇ reduces toxicity, and in mouse models, "reduce[s] ⁇ plaque load and normalizejs] behavioral deficits" (Frost et aL 2012: Wirths et a!., 2010; WO2011 151076: US8283517).
  • C -terminal length e.g., the ⁇ ⁇ -42 species compared to the ⁇ ⁇ -4 ⁇ species
  • N-ierminal length e.g., the ⁇ ⁇ .42 species compared to the Apj. 42 species
  • ⁇ ⁇ ⁇ ...3 ⁇ 42 precedes the deposition of unmodified ⁇ ;, ⁇ in the brain and is more toxic than unmodified ⁇ in AD and Down syndrome (Lemere et aL, 1996; Russo et aL, 2002).
  • transgenic mice expression of ⁇ ⁇ ⁇ 2 causes "massive neurological impairments” starting in early adulthood (Wirths et aL. 2009).
  • transgenic mice that only express unmodified ⁇ exhibit relatively “minimal neurofibrillary pathology and neuronal loss” (Kawarabayashi et aL, 2001).
  • ⁇ 3- ⁇ demonstrates tremendous diagnostic, prognostic, and therapeutic potential
  • detecting and measuring ⁇ 3- ⁇ currently poses many technical challenges.
  • current methods are able to detect ⁇ 3- ⁇ primarily in biological samples containing relatively high levels of ⁇ 3- ⁇ , such as ⁇ plaques and post-mortem AD brains, but detection remains challenging in samples containing relatively low levels of ⁇ 3- ⁇ , such as cerebrospinal fluid.
  • ⁇ 3- ⁇ has been detected in plaques from AD and Down syndrome patients (Frost et aL, 2013) and AD mice ( WO2012021469) by imraunohistochemistry.
  • 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
  • ⁇ , ⁇ 3-42, ⁇ .42, ⁇ 5 - 4 ⁇ , ⁇ 4 ⁇ 2, and Aps- 2 have been detected in murine AD brains by immimopreeipitation combined with mass spectrometry (Wittnam et ah, 2012). ⁇ ). ⁇ , ⁇ , ⁇ ) . 3 9, ⁇ , ⁇ -3 7, ⁇ 2 -4 ⁇ 5 ⁇ 3- 40 ⁇ , ⁇ ⁇ ⁇ 3- ⁇ have been detected in murine AD brains by one- and two-dimensional gel electrophoresis combined with immunobiotting and mass spectrometry (Bibl et ai., 2012). ⁇ ⁇ ⁇ 3-42» ⁇ -42, ⁇ /:.
  • ⁇ plaque formation may be associated with altered levels of ⁇ , ⁇ , and/or ⁇ 3 ⁇ , 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).
  • 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).
  • a highly sensitive method for detecting and measuring ⁇ 3- ⁇ 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.
  • CSF contains particularly low concentrations of ⁇ , 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.
  • highly sensitive assays for detecting and measuring ⁇ 3- ⁇ particularly for biological samples containing low amounts or concentrations of ⁇ 3- ⁇ 5 e.g.. cerebrospinal fluid.
  • the invention provides methods for detecting and measuring amyloid ⁇ in biological samples and diagnostic, prognostic, and therapeutic uses thereof.
  • the invention provides highly sensitive and reliable methods for detecting and measuring ⁇ 3- ⁇ by LC-MS/MS.
  • the methods of the invention may be used to detect and measure ⁇ 3- ⁇ in biological samples containing low levels of pE3 ⁇ ⁇ , such as samples that are very dilute and/or samples of very limited volume.
  • the methods of the invention described herein may be used to detect and measure ⁇ 3- ⁇ in cerebrospinal fluid, in further embodiments, the methods of the invention may be used to distinguish between ⁇ 3- ⁇ peptides of different lengths.
  • the meihods of the invention are used to diagnose and prognose diseases associated with altered levels of ⁇ 3- ⁇ .
  • the meihods of the invention are used to diagnose and prognose diseases associated with altered levels of ⁇ 3- ⁇ .
  • the disease is AD, in particular the early stages of At
  • the methods of the invention may be used to stratify disease slates, monitor disease progression, and/or assess responsiveness to treatment.
  • the methods of the invention may be used to tailor and direct therapies. BRIEF DESCRIPTION OF THE FIGURES
  • 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).
  • LC liquid chromatography
  • FIG, 2 shows a mass spectrum of an ⁇ ⁇ ⁇ 3- 0 full scan.
  • FIG, 3 shows a mass spectrum of an ⁇ ⁇ ⁇ 3- ⁇ M+4H product ion scan.
  • FIG, 4 shows standard curves of A ⁇ - .38, ⁇ -40, ⁇ , ⁇ ,, ⁇ , ⁇ ⁇ 3- ⁇ , and ⁇ 3-42 using solid-phase extraction.
  • a calibration curve of 10 pg/niL - 8 ng/mL is achieved,
  • FIGS. 5A-C show various ⁇ species that were detected in human CSF. Apj . , ⁇ -4 ⁇ , ⁇
  • LOD limit of detection
  • LOQ limit of quantitation
  • FIG. 6 show lysyi endopeptidase (Lys-C) cleavage at the C-terrninus of a lysine residue (K), which is located at position 16 of ⁇ , for ⁇ ⁇ 3-42, ⁇ . 2, amyloid precursor protein.
  • FIG, 7 shows exemplary chromatograms of 20 pg/mL or 2 pg mL of ⁇ ⁇ £. ⁇ 6 and ⁇ .. ) 6 purified by using Lys-C digestion and solid-phase extraction.
  • FIG. 8 shows immunoprecipitation optimization using 150 ⁇ , of 4 pg/mL - 2 ng/mL of ⁇ ⁇ ⁇ 3.] 6 .
  • FIG, 9 shows immunoprecipitation optimization using 2,4 pg mL - 9,6
  • FIG. i O shows the sensitivity and specificity for particular cutoff concentrations of pE3-Aj3 in CSF samples.
  • FIG. 11 demonstrates that the concentration of ⁇ 3- ⁇ in the CvSF of AD patients is significantly lower than the concentration of ⁇ 3 ⁇ in the CSF of control patients
  • MS mass spectrometry
  • 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.
  • 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.
  • 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.
  • 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.
  • a gas e.g., argon, xenon, nitrogen, helium
  • two analyzers are used and the analyzers may be of the same or different types.
  • 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.
  • ESI 'Tdectrospray ionization
  • APCI atmospheric pressure chemical ionization
  • Solid-phase extraction 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”).
  • solid phase a solid through which the solution is passed.
  • 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.
  • Mated 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.
  • 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
  • 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.
  • LC include normal phase liquid chromatography (NPLC), reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance 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.
  • a stationary phase e.g., a densely packed column
  • Selective ion monitoring is a detection mode for a mass spectrometer in which only ions within a relatively narrow mass range are detected.
  • 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.
  • LID Limit of detection
  • 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.
  • purification does not refer to removing al l material from the sample other than the analyte(s) of interest.
  • 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.
  • Amyloid ⁇ and “ ⁇ ” are used interchangeably to refer to amyloid ⁇ peptide and modifications, including pyrogkitamate ⁇ , fragments, and equivalents thereof.
  • ⁇ as used herein refers to any fragment produced by the proteolytic cleavage of amyloid precursor protein (APP).
  • ⁇ oligomers is used to refer to multimeric species of ⁇ that result from the association of monomeric ⁇ species.
  • 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, rheum
  • ⁇ 3- ⁇ or " ⁇ ⁇ as used herein refers to N-terrninally modified pyroglutamate amyloid ⁇ peptides that start at the glutamic acid (Glu) residue at position 3 of the amino acid sequence of ⁇ , wherein the Glu residue is cycfized to form a pyroglutamic acid residue. Examples include but are not limited to ⁇ ⁇ ⁇ 8> ⁇ ⁇ 3 ⁇ 40> arcd ⁇ . 3.42.
  • the amino acid sequences of these N-terminally modified form s of ⁇ peptides are:
  • Unmodified ⁇ ' is used to refer to amyloid ⁇ peptides in which the N terminus is not modified post-trans!aiional iy, including but not limited to ⁇ .38, Ap 0> and ⁇ j -42.
  • the amino acid sequences of these unmodified ⁇ peptides are:
  • An antibody that is "specific for ⁇ 3- ⁇ " 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 ⁇ . including other forms of pyroglutamate ⁇ , e.g., pEl l- ⁇ .
  • Examples of pE3 - ⁇ -specific antibodies include but are not limited to:
  • 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 ALPAPIEKTISKAKGQPREPQVYTLPPSRD
  • Polypeptide ' , '' 'pe tide 1 ', and “protein” are used interchangeably to refer to a polymer of amino acids.
  • the invention provides methods for detecting and measuring ⁇ in biological samples.
  • the invention provides highly sensitive and reliable multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for detecting and measuring ⁇ 3- ⁇ .
  • the methods of the invention may be used for biological samples containing low levels of ⁇ 3 ⁇ 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 ⁇ 3 ⁇ in cerebrospinal fluid.
  • a biological sample that potentially contains pF.3- ⁇ is analyzed by mass spectrometry.
  • 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.
  • fragment Ions are filtered by mass-to-eharge ratio and detected as electronic signals.
  • the mass spectrometry Is tandem mass spectrometry in time or tandem mass spectrometry in space.
  • the methods of the invention are used to diagnose a disease associated with altered levels of ⁇ 3- ⁇ .
  • the methods of the invention are used to prognose a disease associated with altered levels of pE3 ⁇ A[i
  • the methods of the invention are used in the treatment of a disease associated with altered levels of ⁇ 3- ⁇ , for example, by stratifying disease states, monitoring disease progression, and/or assessing responsiveness to treatment,
  • the methods of the invention may be used to tailor and direct therapies.
  • the disease is Alzheimer's disease, in particular in the early stages of the disease.
  • 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.
  • 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.
  • 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
  • the methods of the invention may be used to determine whether one or more subjects exhibit unfavorable concentrations of ⁇ £3- ⁇ , 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,
  • the methods may be used to select, identify, and/or enrich patients for a clinical trial for a disease associated with altered levels of ⁇ 3- ⁇ .
  • the disease is Alzheimer's disease
  • 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.
  • 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.
  • 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.
  • 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.
  • the target of interest in the sample is APP, ⁇ , or ⁇ 3- ⁇ .
  • the target of interest in the sample does not undergo purification.
  • 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).
  • 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”).
  • 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).
  • 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.
  • the SPE step is followed by size exclusion chromatography or SDS-PAGE.
  • the target of interest in the sample is purified by antibody-based sample purification methods.
  • the sample is purified by inimunodepletion, which is intended to extract potential interfering peptides and leave the target of interest in a cleaner medium.
  • 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.
  • 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,
  • the immunocapture is mmvunopreeipitation.
  • at least one pE3 ⁇ Aj3 ⁇ 4 ⁇ specific antibody is used to capture pE3-Aji.
  • 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.
  • the pES-Ap-specific antibody is conjugated to a magnetic bead to form an antibody-bead immunocomplex.
  • the antibody- bead irnmunocompiex binds to and immunoprecipitates ⁇ 3- ⁇ in a biological sample.
  • 3 complex is separated magnetically, and ⁇ 3- ⁇ is eluted.
  • ⁇ 3- ⁇ - ⁇ antibodies include, but are not limited to:
  • R17L (SEQ ID NOs: 7 and 9)
  • the immunoprecipitaiion uses an antibody that is specific for ⁇ 3- ⁇ .
  • the antibody concentration ranges from about 0.1 ⁇ ig/mL to about 2 [ig/mL; about 2 ⁇ / ⁇ to about 5 ⁇ g/mL ⁇ ; or about 5 to about 10 Lig/'mL.
  • pEl l- ⁇ or other ⁇ - ⁇ species could be detected by exchanging the pE3 ⁇ Ap ⁇ specific antibody for a pE1 1 - ⁇ - specific antibody (see, e.g., Perez-Garmendia et al., 2010; commercially available NOP 1-44070) or other ⁇ - ⁇ -specific antibodies.
  • the target of interest in the sample is any substance that is selected from the sample.
  • 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.
  • the target of interest in the sample is denatured and unfolded into single-stranded structures, which are more accessible to the digestion enzyme.
  • the denaturing agent is urea, in some embodiments, the target of interest in the sample is not denatured.
  • 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
  • the reducing agent is dithiothreitol (DTT)
  • the reducing agent is im(2 ⁇ carboxyethyl)phosphine.
  • the target of interest in the sample is not reduced.
  • the peptide is alkylated to prevent the formation of new, non-specific disulfide bonds.
  • the alkylating agent is iodoacetamide.
  • the alkyiation step is performed in dark conditions.
  • the target of interest in the sample is not alkylated.
  • the target of interest in the sample is cleaved by at least one proteolytic enzyme.
  • the target of interest in the sample is cleaved by microwave-assisted formic acid hydrolysis.
  • 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.
  • 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.
  • 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).
  • 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.
  • the separation may increase analysis speed and sample throughput.
  • the target of interest in the sample is separated by liquid chromatography (LC).
  • LC liquid chromatography
  • 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.
  • the particles are semi-porous particles, which are intended to decrease hack pressure.
  • the columns are stationary phase sorbent columns, C8 columns, C I 8 columns, fused core columns, or monolithic columns.
  • the column is an ACE 5 phenyl, 2.1 x 50 mm column,
  • the column temperature is above 35°C. In general, increased column temperature improves separation efficiency.
  • 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
  • the sample is separated by HPLC.
  • HPLC high turbulenc liquid chromatography
  • HTLC high turbulenc liquid chromatography
  • multidimensi nal liquid chromatography in one embodiment, the sample is separated by 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.
  • the methods of the invention are used to determine the concentration or amount of ⁇ 3- ⁇ in a biological sample that, contains low levels of ⁇ 3- ⁇ , such as samples that are very dilute and/or samples of very limited volume.
  • the invention may be used to delect and measure ⁇ 3- ⁇ in cerebrospinal fluid.
  • the methods of the invention may be used to determine whether one or more subjecis exhibit unfavorable concentrations of pE3- ⁇ , 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.
  • 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.
  • the sample is ionized by atmospheric pressure chemical ionization (APCI).
  • APCI atmospheric pressure chemical ionization
  • the sample is ionized by electrospray ionization
  • the detection instrument is a triple quadrupole (QqQ) instrument.
  • the instrument operates in selective reaction monitoring (SRM) mode.
  • the instrument operates in multiple reaction monitoring (MRM) mode.
  • SRM selective reaction monitoring
  • MRM multiple reaction monitoring
  • analyte ions collide with gases in a collision cell (q2) in a collision-induced dissociation step, which further fragments the analyte ions.
  • the gas may be argon, xenon, nitrogen, or helium.
  • 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.
  • the methods of the invention are used to determine the concentration or amount of ⁇ 3- ⁇ 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.
  • at least three additional samples containing known concentrations of ⁇ 3- ⁇ e.g., standards, other biological .samples
  • 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 ⁇ 3- ⁇ .
  • concentrations of ⁇ 3- ⁇ are calculated by comparing the intensity of the ions of the biological sample to the at least three known concentrations of the standard curve.
  • the methods of the invention are used to compare concentrations of ⁇ 3- ⁇ in two or more biological samples. Two or more biological samples are analyzed by MS as described above and concentrations of ⁇ 3- ⁇ 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 ⁇ are lower in AD patients (Mehta et al., 2000: Blennow and Hampel, 2003), possibly due to increased ⁇ aggregation the brain (Fagan et al., 2006; Strozyk et al., 2003), but concentrations of ⁇ 3- ⁇ in the CSF of AD patients were previously unknown.
  • the methods of the invention are used to detect and measure ⁇ 3- ⁇ in CSF samples collected fr m AD patients and healthy controls, which are assayed by MS as described above. Analyses of ⁇ 3 ⁇ in CSF in FIG- 11 demonstrate that the concentration of ⁇ 3- ⁇ in AD patients is significantly lower than the concentration of ⁇ 3- ⁇ in control patients.
  • relative levels of ⁇ 3- ⁇ are determined and compared, in certain embodiments, relative levels of ⁇ 3- ⁇ are determined without using standards and without generating a standard curve.
  • 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 ⁇ 3- ⁇ concentration of the at least one undi agnosed subject may be compared to the ⁇ 3- ⁇ concentration of the at least one AD patient and the at least one healthy control.
  • relative levels of ⁇ 3- ⁇ are measured and compared.
  • the two or more biological samples e.g.. CSF and plasma
  • concentrations of ⁇ 3 ⁇ are measured and compared. It is known, for example, that low concentrations of unmodified ⁇ in the CSF may be indicative of increased ⁇ aggregation in the ⁇ plaques of the brain (Fagan et al.. 2006; Strozyk et al., 2003). in alternate
  • relative levels of ⁇ 3- ⁇ are measured.
  • the methods of the invention are used to detect and measure ⁇ 3- ⁇ after an initial screen for ⁇ 3 ⁇ .
  • the initial screen is an immunoassay, in particular an immunoPCR.
  • the initial screen is an immunoassay, in particular an immunoPCR.
  • the immunoassay detects the presence of ⁇ 3- ⁇ in a biological sample wherein the presence of ⁇ 3- ⁇ is indicative of a disease, and the ⁇ 3- ⁇ concentration is measured by the methods described herein.
  • a second biological sample is collected from a different tissue, and the ⁇ 3- ⁇ concentration is measured by the methods described herein.
  • the methods of the invention are used to detect and measure ⁇ 3- ⁇ before, at the same time as, or after detecting and/or measuring ⁇ 3 ⁇ using the immunoassay.
  • the methods of the invention may be used to detect, but not to measure ⁇ 3- ⁇ .
  • the methods are used to determine the presence or absence of ⁇ 3- ⁇ .
  • the presence of ⁇ 3- ⁇ is determined as a level that is at or above the LOD.
  • the presence of ⁇ 3 ⁇ is determined as a level that is at or above the LOQ.
  • the absence of ⁇ 3- ⁇ Is determined as a level that is below the LOD.
  • the absence of ⁇ 3- ⁇ is determined as a level that is below the LOQ. in biological samples wherein the presence of ⁇ 3- ⁇ is indicative of a disease, the sample is further evaluated by an alternative quantitative method.
  • a second biological sample is collected from a different tissue.
  • the second sample is analyzed for the presence or absence of pE-3- ⁇ , as described above, and/or by an alternative quantitative method.
  • 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 ⁇ 3- ⁇ .
  • the methods of the invention may be used to tailor and direct therapies.
  • the disease is Alzheimer's disease, in particular in the early stages of the disease.
  • the methods of the invention are used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment.
  • 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 ⁇ 3- ⁇ or a fragment thereof, as described herein.
  • the methods may be used to select, identity, and/or enrich patients for a clinical trial for a disease associated with altered levels of ⁇ 3- ⁇ . in certain embodiments, the disease is Alzheimer's disease.
  • 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.
  • 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.
  • the kit is a diagnostic kit.
  • the diagnostic kit may be used for the detection and diagnosis of ⁇ 3- ⁇ diseases and conditions, particularly Alzheimer's disease.
  • a method of detecting ⁇ 3- ⁇ in a biological sample comprising
  • restriction enzyme digestion comprises at least one restriction enzyme that cleaves ⁇ 3- ⁇ into at least one fragment between 3 and 25 amino acids in length.
  • 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.
  • 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.
  • 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
  • HTLC chromatography
  • multidimensional liquid chromatography chromatography
  • a method of diagnosing a disease associated with altered levels of ⁇ 3- ⁇ comprising (a) subjecting a biological sample to ionization to generate one or more charged ions:
  • a method of prognosing a disease associated with altered levels of ⁇ 3- ⁇ comprising
  • a method of developing, tailoring, and/or directing therapies for a disease associated with altered levels of ⁇ 3- ⁇ comprising
  • 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,
  • SAD sporadic Alzheimer's disease
  • FAD
  • any one of embodiments 30-34 to determine whether a subject exhibits unfavorable concentrations of ⁇ 3- ⁇ associated with a disease, wherein the concentration of ⁇ 3- ⁇ 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,
  • kits for assaying a biological sample for the presence, absence, or concentration of ⁇ 3- ⁇ by mass spectrometry comprising (a) instructions for assaying the biological sample for ⁇ 3 ⁇ - ⁇ by mass spectrometry;
  • a method of detecting ⁇ 3- ⁇ in a cerebrospinal fluid sample comprising
  • Alzheimer's amyloid beta-peptide Nat Rev Mo! Celt Biol 8(2): 101 - 12 (2007).
  • ⁇ -!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).
  • 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).
  • Example 1 Internal st ndard, control, and biological samples
  • 6 , ⁇ -30 » and ⁇ -3. 0 were acquired from Anaspec Inc. (Fremont, CA); ⁇ 3 ⁇ 48, ⁇
  • DMSO dimethyl sulfoxide
  • sodiirmphosphate buffer, pH 7.4 (''Buffer B") was added to the tube and vortexed.
  • the tube was placed on a DynaMagTM-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 DynaMagTM-2 magnet for 1 minute, and the supernatant was removed.
  • the magnetic beads were resuspended in 100 pL of Buffer B.
  • Buffer E 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 DynaMagTM ⁇ 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.
  • 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 DynaMagTM-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 DynaMagTM-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.
  • 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.
  • Each calibration standard or QC for ⁇ 3- ⁇ was prepared by adding 15 pL of spiking solution of ⁇ .3- 0 and 15 pL internal standard spiking solution of heavy ⁇
  • 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.
  • 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.
  • 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 DynaMagTM-2 magnet for 1 minute, and the supernatant was removed.
  • 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 DynaMagTM-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
  • 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
  • the detector was a Waters Xevo TQ-S triple quadrupole mass
  • Ta tble 2 MR M transitions for pE3 m y!oid beta assay
  • Nebuliser : 7.0 Bar
  • 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).
  • 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% H 3 PO 4 , 800 pL of the pre -treated biological samples, described above, were added to the plate.
  • the plate was washed with 600 ⁇ , of 0.5% H3PO4; 400 ⁇ , of 0.4% H3PO4 and 40% acetonitrile; 400 pL of 0.4% H 3 PO 4 and 80% acetonitrile; and 600 ⁇ of water.
  • FIG. 4 shows standard curves of ⁇
  • a calibration curve of 10 pg/mL - 8 ng/mL is achieved.
  • FIGS. 5A-C show particular ⁇ species that were detected in human CSF. ⁇ .42, ⁇ -4 , ⁇ -38 were detected in pooled human CSF without using solid-phase extraction (FIG. 5A).
  • Biological samples were digested using a restriction enzyme prior to solid-phase extraction (SPE). 200 pL of a biological sample was incubated with 200 ⁇ ., of 50 m Tris, 2 mM EDTA, pH 8.5, and 20 M L of 100 ⁇ , Wake lysyl
  • Lys-C endopeptidase 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 ⁇ (FIG. 6),
  • 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 ⁇ , of the digested samples, described above, were added to the plate. The plate was washed with 600 ⁇ , of 0.5% H 3 P0 4 ; 300 uL of 0.4% H3PO4 and 40% acetomtrile; 300 ⁇ , of 0,4% i i 3 P(> 4 and 80% acetonitrile; and 600 pL of water.
  • FIG. 7 shows chromatograrns of 20 pg/mL (45 pL) of ⁇ ⁇ ⁇ 3- ⁇ 6> 20 pg/mL (45 ⁇ ,) of ⁇
  • Exam le 9 LC-MS/MS analysis with Lys ⁇ C digestion, solid-phase extraction, mid isHMMO reci itaiioa
  • Biological samples containing 200 pg/mL of ⁇ ⁇ ⁇ 3-40 were digested using the restriction enzyme Lys-C prior to solid-phase extraction, as described above.
  • ⁇ ⁇ ⁇ 3-; in the sample was captured by magnetic Dynabeads® conjugated to ABl 00-1 1 . as described above.
  • the sample was washed, and Ap pE 3.,': 6 -beacl complex was isolated.
  • the ⁇ ⁇ ⁇ 3-! ⁇ was released from the Dynaheads® by heat, acidification, and organic solvent washing.
  • CSF containing 1 50 ⁇ ... of 4 pg/mL - 2 ng/mL of of ⁇ ⁇ 3 - ⁇ 6 ( FIG. 8), 2 pg/mL - 20 pg/mL ⁇ ⁇ ⁇ 3- ⁇ 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 ⁇ ⁇ .4 0 (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
  • the methods of the invention may be used to detect and measure ⁇ 3- ⁇ in cerebrospinal fluid, which is challenging using current methods, ⁇ 3- ⁇ in CSF samples was detected and measured using Lys-C digestion, SPIT immunoprecipitation, and mass spectrometry, as described above.
  • ⁇ ⁇ ⁇ 3- 0 was detected in individual CSF samples (Table 5).
  • the limit of detection (LOD) for ⁇ ⁇ ⁇ 3- ⁇ was 0.33 pg mL and the limit of quantitation (LOQ) for ⁇ ⁇ - 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

Landscapes

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

Abstract

L'invention concerne des méthodes permettant de détecter et de mesurer pΕ3-Αβ et des utilisations des méthodes pour diagnostiquer et/ou pronostiquer des maladies associées à des niveaux modifiés de pΕ3-Αβ. L'invention porte également sur des utilisations des méthodes pour mettre au point, adapter spécifiquement et mener des thérapies.
PCT/US2015/035282 2014-06-13 2015-06-11 Méthodes pour la détection et la mesure de la protéine beta amyloïde dans des échantillons biologiques WO2015191825A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462011960P 2014-06-13 2014-06-13
US62/011,960 2014-06-13

Publications (1)

Publication Number Publication Date
WO2015191825A1 true WO2015191825A1 (fr) 2015-12-17

Family

ID=53491677

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/035282 WO2015191825A1 (fr) 2014-06-13 2015-06-11 Méthodes pour la détection et la mesure de la protéine beta amyloïde dans des échantillons biologiques

Country Status (1)

Country Link
WO (1) WO2015191825A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9828420B2 (en) 2007-01-05 2017-11-28 University Of Zürich Method of providing disease-specific binding molecules and targets
WO2018083628A1 (fr) * 2016-11-03 2018-05-11 Janssen Pharmaceutica Nv ANTICORPS ANTI-PYROGLUTAMATE- AMYLOÏDE β ET LEURS UTILISATIONS
US10842871B2 (en) 2014-12-02 2020-11-24 Biogen International Neuroscience Gmbh Methods for treating Alzheimer's disease
JP2021059546A (ja) * 2016-07-01 2021-04-15 イーライ リリー アンド カンパニー 抗Ｎ３ｐＧｌｕアミロイドベータペプチド抗体およびその使用
CN113527459A (zh) * 2021-06-28 2021-10-22 广州睿徕医学诊断技术有限公司 一种萃取剂及其制备方法与应用
CN113848258A (zh) * 2020-06-25 2021-12-28 希森美康株式会社 Aβ肽的测定方法及在该方法中使用的试剂组合物
US11236155B2 (en) 2019-03-26 2022-02-01 Janssen Pharmaceutica Nv Antibodies to pyroglutamate amyloid-β and uses thereof
US11655289B2 (en) 2017-08-22 2023-05-23 Biogen Ma Inc. Pharmaceutical compositions containing anti-beta amyloid antibodies

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050260697A1 (en) * 2004-04-15 2005-11-24 University Of Florida Research Foundation, Inc. Proteolytic markers as diagnostic biomarkers for cancer, organ injury and muscle rehabilitation/exercise overtraining
JP2008309778A (ja) * 2007-05-11 2008-12-25 Daiichi Sankyo Co Ltd ポリペプチドの検出又は定量方法、及び装置
WO2011064225A1 (fr) * 2009-11-24 2011-06-03 Probiodrug Ag Nouveau procédé de diagnostic pour le diagnostic de la maladie d'alzheimer ou d'un trouble cognitif léger
US20110152341A1 (en) * 2009-12-22 2011-06-23 Probiodrug Ag Cleavage of b-amyloid precursor protein
WO2012005838A1 (fr) * 2010-07-07 2012-01-12 Thermo Fisher Scientific Gmbh Quantification d'analytes en spectrométrie de masse à l'aide d'un indicateur universel

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050260697A1 (en) * 2004-04-15 2005-11-24 University Of Florida Research Foundation, Inc. Proteolytic markers as diagnostic biomarkers for cancer, organ injury and muscle rehabilitation/exercise overtraining
JP2008309778A (ja) * 2007-05-11 2008-12-25 Daiichi Sankyo Co Ltd ポリペプチドの検出又は定量方法、及び装置
WO2011064225A1 (fr) * 2009-11-24 2011-06-03 Probiodrug Ag Nouveau procédé de diagnostic pour le diagnostic de la maladie d'alzheimer ou d'un trouble cognitif léger
US20110152341A1 (en) * 2009-12-22 2011-06-23 Probiodrug Ag Cleavage of b-amyloid precursor protein
WO2012005838A1 (fr) * 2010-07-07 2012-01-12 Thermo Fisher Scientific Gmbh Quantification d'analytes en spectrométrie de masse à l'aide d'un indicateur universel

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE Geneseq [online] 21 July 2011 (2011-07-21), "Human amyloid beta protein fragment (3-38), SEQ ID 13.", XP002743933, retrieved from EBI accession no. GSP:AZI67190 Database accession no. AZI67190 *
ERIC SHUANG PANG: "Mass Spectrometry-Based Structural Probing of Amyloid /3-Protein", 2011, XP055210953, Retrieved from the Internet <URL:http://media.proquest.com/media/pq/classic/doc/2594153351/fmt/ai/rep/SPDF?hl=&cit:auth=Pang, Eric Shuang&cit:title=Mass Spectrometry-Based Structural Probing of Amyloid beta-Protein&cit:pub=ProQuest Dissertations and Theses&cit:vol=&cit:iss=&cit:pg=n/a&cit:date=2011&ic=true&cit:prod=ProQuest Dissert> [retrieved on 20150903] *
ERIK PORTELIUS ET AL: "Characterization of Amyloid [beta] Peptides in Cerebrospinal Fluid by an Automated Immunoprecipitation Procedure Followed by Mass Spectrometry", JOURNAL OF PROTEOME RESEARCH, vol. 6, no. 11, 1 November 2007 (2007-11-01), pages 4433 - 4439, XP055041397, ISSN: 1535-3893, DOI: 10.1021/pr0703627 *
GELFANOVA VALENTINA ET AL: "Quantitative analysis of amyloid-beta peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry", BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS, HENRY STEWART, LONDON, GB, vol. 6, no. 2, 1 June 2007 (2007-06-01), pages 149 - 158, XP002607984, ISSN: 1473-9550 *
J. L. WITTNAM ET AL: "Pyroglutamate Amyloid (A ) Aggravates Behavioral Deficits in Transgenic Amyloid Mouse Model for Alzheimer Disease", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, no. 11, 9 March 2012 (2012-03-09), pages 8154 - 8162, XP055210579, ISSN: 0021-9258, DOI: 10.1074/jbc.M111.308601 *
LEWCZUK P ET AL: "Amyloid beta peptides in cerebrospinal fluid as profiled with surface enhanced laser desorption/ionization time-of-flight mass spectrometry: evidence of novel biomarkers in Alzheimer's disease", BIOLOGICAL PSYCHIATRY, ELSEVIER SCIENCE, NEW YORK, NY; US, vol. 55, no. 5, 1 March 2004 (2004-03-01), pages 524 - 530, XP027164177, ISSN: 0006-3223, [retrieved on 20040223] *
PORTELIUS ERIK ET AL: "Determination of beta-amyloid peptide signatures in cerebrospinal fluid using immunoprecipitation-mass spectrometry", JOURNAL OF PROTEOME RESEARCH, ACS, WASHINGTON, DC, US, vol. 5, no. 4, 1 April 2006 (2006-04-01), pages 1010 - 1016, XP002550741, ISSN: 1535-3893, [retrieved on 20060310], DOI: 10.1021/PR050475V *
S. JAWHAR ET AL: "Pyroglutamate Amyloid- (A ): A Hatchet Man in Alzheimer Disease", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 286, no. 45, 11 November 2011 (2011-11-11), pages 38825 - 38832, XP055086822, ISSN: 0021-9258, DOI: 10.1074/jbc.R111.288308 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10202445B2 (en) 2007-01-05 2019-02-12 University Of Zurich Method of providing disease-specific binding molecules and targets
US9828420B2 (en) 2007-01-05 2017-11-28 University Of Zürich Method of providing disease-specific binding molecules and targets
US10131708B2 (en) 2007-01-05 2018-11-20 University Of Zürich Methods of treating Alzheimer's disease
US10842871B2 (en) 2014-12-02 2020-11-24 Biogen International Neuroscience Gmbh Methods for treating Alzheimer's disease
JP2021059546A (ja) * 2016-07-01 2021-04-15 イーライ リリー アンド カンパニー 抗Ｎ３ｐＧｌｕアミロイドベータペプチド抗体およびその使用
JP7241058B2 (ja) 2016-07-01 2023-03-16 イーライ リリー アンド カンパニー 抗Ｎ３ｐＧｌｕアミロイドベータペプチド抗体およびその使用
CN109922826A (zh) * 2016-11-03 2019-06-21 詹森药业有限公司 焦谷氨酸淀粉样蛋白-β的抗体及其用途
US10519223B2 (en) 2016-11-03 2019-12-31 Jannsen Pharmaceutica Nv Antibodies to pyroglutamate amyloid-β and uses thereof
WO2018083628A1 (fr) * 2016-11-03 2018-05-11 Janssen Pharmaceutica Nv ANTICORPS ANTI-PYROGLUTAMATE- AMYLOÏDE β ET LEURS UTILISATIONS
US10851156B2 (en) 2016-11-03 2020-12-01 Janssen Pharmaceutica Nv Methods of detecting pyroglutamate amyloid beta protein (3pE Aβ) using anti-3pE Aβ antibodies
US11673943B2 (en) 2016-11-03 2023-06-13 Janssen Pharmaceutica Nv Methods of binding amyloid beta protein having pyroglutamate at the third amino acid residue (3pE abeta) in vivo using anti-3pE abeta antibodies or antigen-binding fragments thereof
US11655289B2 (en) 2017-08-22 2023-05-23 Biogen Ma Inc. Pharmaceutical compositions containing anti-beta amyloid antibodies
US11236155B2 (en) 2019-03-26 2022-02-01 Janssen Pharmaceutica Nv Antibodies to pyroglutamate amyloid-β and uses thereof
CN113848258A (zh) * 2020-06-25 2021-12-28 希森美康株式会社 Aβ肽的测定方法及在该方法中使用的试剂组合物
CN113527459A (zh) * 2021-06-28 2021-10-22 广州睿徕医学诊断技术有限公司 一种萃取剂及其制备方法与应用

Similar Documents

Publication Publication Date Title
JP7008268B2 (ja) 脳内のアミロイドβペプチド蓄積状態を評価するサロゲート・バイオマーカー及びその分析方法
WO2015191825A1 (fr) Méthodes pour la détection et la mesure de la protéine beta amyloïde dans des échantillons biologiques
Barthélemy et al. Tau phosphorylation rates measured by mass spectrometry differ in the intracellular brain vs. extracellular cerebrospinal fluid compartments and are differentially affected by Alzheimer’s disease
US20080220449A1 (en) Biomarkers and assays for Alzheimer&#39;s disease
Wijte et al. A novel peptidomics approach to detect markers of Alzheimer’s disease in cerebrospinal fluid
Höglund et al. Cerebrospinal fluid neurogranin in an inducible mouse model of neurodegeneration: A translatable marker of synaptic degeneration
Rudinskiy et al. Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint
US20020006627A1 (en) Method for diagnosis of Alzheimer&#39;s disease
WO2010032458A1 (fr) Biomarqueur inédit de la stéatose hépatique non alcoolique et méthode de détection d&#39;une stéatose hépatique non alcoolique au moyen dudit biomarqueur
Guy et al. The impact of antibody selection on the detection of cardiac troponin I
WO2011094645A1 (fr) Méthodes de diagnostic de maladies neurodégénératives associées à tau
JP5488885B2 (ja) 感染症の疾患マーカー
JP2024028781A (ja) アルツハイマー病の診断と予後判定におけるマーカーとしてのｐ５３ペプチド
Pobran et al. Aptamer-based enrichment of TDP-43 from human cells and tissues with quantification by HPLC-MS/MS
JP7215903B2 (ja) タンパク質の構造型の検出
Rezeli et al. MRM assay for quantitation of complement components in human blood plasma—a feasibility study on multiple sclerosis
JP6445861B2 (ja) オキシトシン検出のためのサンプルの前処理方法
WO2007140971A2 (fr) Protéines d&#39;adhérence cellulaire utilisées en tant que biomarqueurs de la maladie d&#39;alzheimer
EP4375661A1 (fr) BIOMARQUEUR D&#39;Aß EN SOURIS DE MODÈLE DE MALADIE D&#39;ALZHEIMER (MA) ET MÉTHODE ASSOCIÉE D&#39;ANALYSE
AU2003274368B2 (en) Diagnostic method for transmissible spongiform encephalopathies (prion diseases)
Lantero Rodriguez Novel cerebrospinal fluid and blood tau biomarkers in Alzheimer's disease and other neurodegenerative diseases
JP2023536162A (ja) 神経変性疾患の診断と予後推定におけるマーカーとしてのｐ５３の翻訳後修飾
O'Brien The Development and Implementation of a TMT-SRM Assay for the Validation of Candidate Biomarkers of Alzheimer's Disease
CA2373150A1 (fr) Procede de diagnostic de la maladie d&#39;alzheimer
JP2020064051A (ja) 神経変性疾患の診断用ペプチドマーカー

Legal Events

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

Ref document number: 15732490

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15732490

Country of ref document: EP

Kind code of ref document: A1