Analyzing oligomeric protein structures by mass spectrometry

Abstract

Disclosed herein are methods for analyzing oligomeric protein structures by mass spectrometry. The method includes providing a sample having one or more oligomers; producing, with an ion source, ions of the sample, each of the ions having a mass-to-charge (m z) ratio; detecting a multiplicity of ions generated with a current detector; determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer; generating a mass-domain spectrum from the ion masses with the mass-analyzer, the mass-domain spectrum having one or more mass-domain peaks; and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers. Methods for diagnosing a subject, assessing treatment efficacy, and assessing treatment efficacy are also provided.

Description

[0001] ANALYZING OLIGOMERIC PROTEIN STRUCTURES BY MASS SPECTROMETRY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority to U.S. Patent Application Ser. No. 63 / 608,700, filed December 11, 2023. The contents to which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant numbers AG069456, AG063903 and DA018310 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Amyloid fibrils are a common thread through a myriad of neurodegenerative diseases, including Alzheimer’s Disease (AD) and Parkinson’s Disease. In the case of AD, tau (~55 kDa) and amyloid beta (~4.5 kDa) are known aggregators that have been labeled as possible causative biomarkers. As such, they have been subjects of intense study and debate in pursuit of more effective diagnostic and treatment techniques. However, focus has shifted away from fibrils and towards soluble oligomers in recent years. These intermediates between individual building blocks and fibrils have been identified as highly toxic and a candidate driver of neurodegeneration. However, the heterogeneity of endogenous oligomers is an immense barrier for analysis. MS detects components on the basis of mass-to-charge ratio (m z) on top of any preexisting heterogeneity, of which one mass value ( i) may exhibit many charge states (z). Therefore, even small oligomers (< 100 kDa) are unintelligible by normal means. Charge detection MS (CDMS) offers a means of spectral decongestion by measuring ion charge, placing analytes directly on the mass domain instead of the m / z domain. CDMS has been used to measure the composition of synthetic amyloid fibrils (including amyloid beta fibrils), but these studies utilized low-resolution ion trap mass spectrometers and did not provide any information on oligomeric intermediates.

[0008] Therefore, there is a need in the field for high resolution, high-throughput methods for characterizing intermediate oligomers. BRIEF SUMMARY OF THE INVENTION

[0009] Disclosed herein are methods for analyzing oligomeric protein structures by mass spectrometry. The method includes providing a sample having one or more oligomers; producing, with an ion source, ions of the sample, each of the ions having a mass-to-charge (m z) ratio; detecting a multiplicity of ions generated with a current detector; determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer; generating a mass-domain spectrum from the ion masses with the mass-analyzer, the mass-domain spectrum having one or more mass-domain peaks; and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers.

[0010] Another aspect of the technology provides for a method for diagnosing a subject suspected of needing a treatment for an oligomer-associated disease or condition. The method may comprise providing a sample suspected of having one or more oligomers from the subject; producing, with an ion source, ions of the sample, each of the ions having a mass-to-charge (m / z) ratio; detecting a multiplicity of ions generated with a current detector; determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer; generating a massdomain spectrum from the ion masses with the mass-analyzer, the mass-domain spectrum having one or more mass-domain peaks; determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers; and comparing the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers to one or more diagnostic reference metrics of an oligomer-associated condition.

[0011] Another aspect of the technology provides for a method for assessing efficacy of treatment of a subject having an oligomer-associated disease or condition. The method may comprise providing a pre-treatment sample containing one or more oligomers from the subject before treatment is administered; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the pre-treatment sample according to methods disclosed herein; administering one or more treatments to the subject; providing a post-treatment sample containing one or more oligomers from the subject after the one or more treatments are administered; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the post-treatment sample according to methods disclosed herein; and comparing the one or more metrics capturing the heterogeneity and / or abundance of oligomers in the pre-treatment sample to the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers in the post-treatment sample.

[0012] Another aspect of the technology provides for a method for assessing progression of an oligomer-associated disease or condition in a subject. The method may comprise providing a first sample containing one or more oligomers from the subject; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the first sample according to a method disclosed herein; providing a second sample obtained after the first sample is obtained from the subject containing one or more oligomers; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the second sample according to a method disclosed herein; andbcomparing the one or more metrics capturing the heterogeneity and / or abundance of oligomers in the first sample to the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers in the second sample.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0015] FIG. 1 shows the measurement of amyloid beta oligomers via I2MS. Panel (a) shows conventional m / z spectra of oligomers derived from 5xFAD mouse models (using the antibodies 4G8 and NU2) and synthesis. Panel (b) shows I2MS processed version of the data from panel (a), in the mass domain. The dotted blue line marks 55 kDa, the approximate expected molecular weight of a dodecamer from canonical A[3(l-42), which is a pivotal stoichiometry in the oligomerization mechanism. The enlarged region depicts the molecular weight range lower than a dodecamer in size. Panel (c) shows heat maps depicting the ion data from panel (b) in terms of assigned charge. Cells with zero ions were colored white to enhance clarity.

[0016] FIG. 2 shows peak analysis of amyloid beta oligomer mass spectra. Panel (a) shows mass scatterplot of peak centroids within the context of their overall peak series. Smooth lines are shown to highlight nonlinearity. Panel (b) shows Fourier transforms of subregions of each mass spectra (i.e., 12-55 kDa and 55-165 kDa). Coherent signal on the 1 / Frequency domain denotes consistent peak spacings of that value, corresponding to a recurring building block (e.g., amyloid beta monomer, dimer, or trimer).

[0017] FIG. 3 shows a complete data set (both replicates) of the measurement of amyloid beta oligomers via I2MS. Panel (a) shows conventional m / z spectra of oligomers derived from 5xFAD mouse models (using the antibodies 4G8 and NU2) and synthesis. Panel (b) shows I2MS processed version of the data from panel (a), in the mass domain. The enlarged region depicts the molecular weight range lower than a dodecamer in size. Panel (c) shows heat maps depicting the ion data from panel (b) in terms of assigned charge. Cells with zero ions were colored white to enhance clarity.

[0018] FIG. 4 shows a Complete data set of the peak analysis of amyloid beta oligomer mass spectra. Panel (a) shows mass scatterplot of peak centroids within the context of their overall peak series. Smooth lines are shown to highlight nonlinearity. Panel (b) shows Fourier transforms of subregions of each mass spectra (i.e., 12-55 kDa and 55-165 kDa). Coherent signal on the 1 / Frequency domain denotes consistent peak spacings of that value, corresponding to a recurring building block (e.g., amyloid beta monomer, dimer, or trimer).

[0019] FIG. 5 shows a single m / z acquisition for all three samples: 4G8, NU2, and synthetic.

[0020] FIG. 6 shows cryogenic transition electron microscopy of NU2-purified amyloid beta oligomers. Panel (a) shows a collection of oligomers. Panel (b) shows a collection of proto-fibrils. Panel (c) shows an example of an oligomer seeding off of a proto-fibril. Copper grids with 300 mesh and a carbon film (EMS Cat. No. CF300-Cu-50) were glow discharged in a Pelco easiGlow glow discharger (Ted Pella Cat. No. 91040) for 20 seconds at 0.26 mBar in a 15 mA atmosphere plasma in order to allow sample to spread evenly on the otherwise hydrophilic carbon surface. Thirty seconds after drop-casting 5 pL of sample on the grid, the droplet was wicked away with filter paper, but the grid was not allowed to dry prior to applying 5 pL of 1% Uranyl Acetate (mixed from stock powder (SPI Cat. No. 02624-AB)) as a negative stain. The negative stain droplet was wicked up and again replaced three times without the grid being allowed to dry. The final droplet of negative stain sat in place on the grid for four minutes before wicking it off with filter paper; the grid was given >4minutes to dry before being loaded into a Hitachi HD2300 cFEG Scanning Transmission Electron Microscope for viewing. Image data was gathered with a Digiscan system within Gatan Digital Micrograph software while utilizing the high angle annular dark field and transmission phase contrast detectors within the microscope.

[0021] FIG. 7 shows average numbers of raw and charge-assigned ions per acquisition across experiments, where each experiment had 3600 acquisitions.

[0022] FIG. 8 shows charge-versus-mass heat map of four standard proteins in native mode, to show the lack of mass heterogeneity for standard proteins.

[0023] FIG. 9 shows oligomer abundance and size distribution in 5xFAD mouse brains. Aqueous extracts were prepared from 15-month-old mouse brain tissue (cortex and hippocampus). ApOs levels were assessed by sandwich ELISA using oligomer-selective antibodies m93 and NUscl. Either non-fractionated samples (a) or filtrates and retentates from a molecular weight based- centrifugal filtration (Amicon) (using the 5xFAD extract as input (b) were assayed. Note that a significant amount of A Os is detected in the 100 kDa retentate fraction (dashed orange, 100 kDa MWCO), while only a small proportion is found in the lower molecular weight fraction (solid orange, 30 kDa MWCO). Images from representative wells corresponding to non-fractionated WT and 5xFAD extracts after signal development are shown at the top of the bars in (a). A scheme of the filtration device used and corresponding MW fractions is depicted in (b). Total protein levels in each sample were measured and normalized before filtration.

[0024] FIG. 10 show relative quantification of ApOs immunoprecipated by NU2 from WT and 5xFAD mouse brain extracts via ELISA and dot blot. The picture on top (a) depicts a representative well from each group after assay development. Brain extracts were incubated with NU2- conjugated beads, and bound ApOs were eluted using ammonium hydroxide in water (see Materials and Methods in the main text). Eluted ApOs from 5xFAD extracts could be detected by both ELISA (a) and dot blot (b). Each point corresponds to a technical replicate from extracts prepared using tissue from one animal / genotype. In (b), dilutions of eluates used in the dot blot are indicated, as well as signal obtained using increasing concentrations of synthetic ApOs.

[0025] FIG. 11 shows Relative quantification of ApOs immunoprecipated by NU2 from a second pair of WT and 5xFAD mouse brain extracts via ELISA, (a) ApOs are abundant in brain extracts from 15-month-old 5xFAD, as detected by ELISA. Brain extracts were incubated with NU2- funcionalized beads, and bound ApOs were released using basic solution. Released ApOs from 5xFAD extracts could be detected by ELISA (b). Each point corresponds to a technical replicate from two independent extracts prepared using tissue from one animal / genotype. Asterisk denotes p-value < 0.05 (t test).

[0026] FIG. 12 illustrates a method according to the technology disclosed herein. Dotted lines indicate optional steps.

[0027] FIG. 13 illustrates a method according to the technology disclosed herein. Dotted lines indicate optional steps.

[0028] FIG. 14 illustrates a method for assessing efficacy of a treatment of a subject in need according to the technology disclosed herein. Dotted lines indicate optional steps.

[0029] FIG. 15 illustrates a method for assessing disease progression in a subject. Dotted lines indicate optional steps.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] Disclosed herein are methods for characterizing oligomers by individual ion mass spectrometry. The methods disclosed allow for the characterization of oligomers by peak spacing analysis in each of the mass spectrum and mass frequency spectrum, making mass heterogeneity and mass frequency heterogeneity of the sample apparent. The analysis of oligomers by the methods disclosed herein additionally may enable methods of diagnosing subjects suspected of having or needing a treatment for an oligomer-associated disease or condition, assessing efficacy of treatment, or assessing progression of an oligomer associated disease or condition.

[0032] Oligomer, as used herein, refers to substances composed of two or more protein building blocks. The proteins contained within the oligomer may be the same or different. Differences between proteins may include differences in the amino acid structure, e.g., differences in the primary, secondary, tertiary, or quaternary structure, and / or post-translational modifications of the proteins. The oligomers may include one or more different proteoforms. As used herein, "proteoform" refers to all of the different molecular forms in which the protein product of a single gene can be found, including isoforms, alternatively spliced RNA transcripts, posttranslational modifications, site-specific features, changes due to genetic variations, etc. As distinct proteoforms can have distinct functions in biology, understanding oligomers within the context of their diverse proteoform components is essential. Oligomers may include between 2 and about 100 proteins, 2 and about 50 proteins, 2 and about 40 proteins, 2 and about 30 proteins, or 2 and about 20 proteins. Suitably, oligomers may be composed of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 proteins. A sample containing oligomers may be synthetic or derived from a biological source, such as an in vivo, in vitro, or ex vivo source. This may include an endogenous oligomer or endogenous oligomeric protein. In some cases, the sample may include one or more cells, one or more tissues, one or more organs, one or more bodily fluids (e.g., cerebrospinal fluid, blood, plasma, serum, lymph, saliva, mucus, urine, and the like)or any combinations thereof. The sample may comprise a mixture of different oligomers. In some cases, the source of the sample may be processed in order to supply a sample with desirable sample handling features. For example, the tissue may be homogenized with a buffer in order to produce a suspension of cells, cell fragments, cell constituents, and any combinations thereof.

[0033] In some cases, the sample may be supplied by a subject. As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal. In some embodiments, the subject is a subject in need of treatment, or a subject suspected to be in need of treatment. A “subject in need of treatment” may include a subject having a disease, disorder, or condition that is associated with, correlated with, or caused by oligomeric proteins. For example, a “subject in need of treatment” may include a subject having a neurodegenerative disease associated with oligomers, such as amyloid beta oligomeric proteins associated with Alzheimer’s disease. A ‘subject suspected to be in need of treatment’ may include a subject who does not have a confirmed disease, disorder, or condition associated with, correlated with, or caused by oligomeric proteins, but may possess or exhibit an oligomer of interest. In some cases, an oligomer of interest may include oligomers whose presence, population, or composition may be indicative of health status.

[0034] Amyloid fibrils are a common thread through a myriad of neurodegenerative diseases, including Alzheimer’s Disease (AD) and Parkinson’s Disease. These noncovalent structures, sometimes consisting of similar or identical protein building blocks, can reach hundreds or thousands of megadalton (MDa) in size and form insoluble deposits in the brain. In the case of AD, tau (~55 kDa) and amyloid beta (~4.5 kDa) are known aggregators and possible causative biomarkers. As such, they have been subjects of intense scrutiny in pursuit of more effective AD diagnostic and treatment techniques.

[0035] Soluble oligomers, such as dodecamers in the case of amyloid beta (“A ”), have garnered attention. Oligomers may be highly toxic intermediates between individual building blocks and fibrils and are candidate drivers of neurodegeneration. The soluble nature of these structures is problematic, as they require targeted purification from other soluble components in biological systems such as the 5xFAD A0 mouse model. Oligomer generation in vitro is viable using synthetic components, but such systems are strongly limited in scope due to their components’ homogeneity — typically A0(l-42) or A0(1-4O) in the case of amyloid beta. For example, Wildburger et al., identified 26 amyloid beta “proteoforms” in select human AD brain tissue, of which canonical A0(l-42) only comprises -15%. [Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain. Sci Rep 2017, 7, 9520] As such, the field has pursued antibodies that selectively target soluble oligomers, such as NU2 in the case of A0.

[0036] Isolated oligomers may be candidates for analysis by mass spectrometry (MS). This method is capable of not only high-resolution proteoform characterization en masse but also characterization of “multi-proteoform complexes” (MPCs), a category which includes oligomers. However, the heterogeneity of endogenous oligomers is an immense barrier for analysis. MS detects components in terms of mass-to-charge ratio (m / z on top of any preexisting component heterogeneity. Component heterogeneity can be approximated through basic combinatorics: tn + k — 1\ n choose k, with replacement, disregarding order = 1 I (1)

[0037] \ K /

[0038] When the 26 candidate proteoforms (n) from Wildburger et al. are considered for a dodecamer (k), Equation 1 amounts to -1.9 billion combinations at 25-54 kDa. Therefore, even populations of small oligomers (<100 kDa) are presumably unintelligible by normal means, MS or otherwise. This approximation compounds with evidence by prior matrix-assisted laser desorption / ionization data that suggests even oligomers of pure A0(l-42) can become poorly resolved at moderate stoichiometries (-10). It is possible to blindly eject monomeric components from oligomers via collisions with neutral gases (e.g., nitrogen), but this method provides only indirect information about the intact oligomeric structures.

[0039] The disclosed technology allows for probing and understanding endogenous oligomers. Differences in oligomeric distributions can be traced through not only mass differences but also spacing differences, allowing for in insights in the underlying biology that may can be associated with research endeavors, diagnosing subjects, assessing treatment efficacy, or assessing disease progression. Referring to Figure 12, a method for oligomer characterization 100 includes providing a sample having one or more oligomers 99, producing ions of the sample with an ion source where each of the ions having a mass-to-charge (m / z) ratio 101, detecting a multiplicity of ions generated with a current detector 102, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 103, generating a mass-domain spectrum from the ion masses with the mass-analyzer 104, the mass-domain spectrum having one or more massdomain peaks, and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 105. Methods for determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers comprises may comprise identifying mass frequencies by applying one or more Fourier transforms to the mass spectra. In some cases, applying a Fourier transform may include applying a sliding window Fourier transform or a Fourier Transform to a sub-region of interest in the mass spectrum.

[0040] Optionally, oligomers may be purified, isolated, or concentrated 1001 before subsequent ionization and analysis. Affinity purification, immunopurification, and other methods already known in the art may be used to purify, isolate, or concentrate oligomers. In some cases, immunopurification using antibody conjugated surfaces (i.e., beads) may be used to selectively target one or more oligomers of interest. Alternatively, membrane-based filtering may be used to select for a given size range of oligomers or concentrate in solution, chromatography (e.g., sizeexclusion chromatography) may be used to select or fractionate samples, or electrophoresis (e.g., gel- or capillary-based electrophoresis) may be used to separate populations of oligomers.

[0041] The method for characterizing an oligomer comprises ionizing a sample with an ionizer and detecting a multiplicity of ions generated by the ionization of the sample with a current detector. In some embodiments, the ionizer and the current detector are components of a mass spectrometer, as already known in the art. From the detected ions, the mass of each of the ions may be determined with a mass analyzer. As used herein, "mass analyzer" may include a programmable processor or combination of processors, such as central processing units (CPUs), graphics processing units (GPUs), Field Programmable Gate Arrays (FPGAs), Application-Specific Integrated Circuits (ASICs) and the like. As such, the mass analyzer may be configured to execute instructions stored in a non-transitory computer readable-media. In this regard, the mass analyzer may be a computer, workstation, laptop or other general-purpose computing device. Additionally or alternatively, the controller may also include one or more dedicated processing units or modules that may be configured (e.g. hardwired, or pre-programmed) to carry out steps, in accordance with aspects of the present disclosure. The mass analyzer is configured to receive a signal from the current detector to determine the ion masses and generate a mass-domain spectrum. Mass spectrometry has used ions to measure the mass-to-charge (m / z) ratio of molecules once lifted into the gas phase. Denatured and native electrospray ionization of intact oligomeric proteins and their complexes pose many complications due to sample heterogeneity and large charge-state envelopes in the m / z domain. Several different methods may be utilized to generate a mass-domain spectrum. To simplify analysis, charge detection mass spectrometry (CDMS) may be used to generate a true mass spectra with the direct readout of an ion’s integer charge value. An exemplary approach for determining the ion masses and generating a mass-domain spectrum includes Orbitrap CDMS, such as individual ion mass spectrometry (I2MS) or Direct Mass Technology Mode (DMTm), ion-trap CDMS, or Fourier- transform ion cyclotron resonance (FTICR) CDMS.

[0042] I2MS allows for measuring oligomers or complex proteoform mixtures and their complexes without the need to separate the proteoforms prior to identification. Suitable methods and systems for I2MS are disclosed in Kafader, J.O. Nat Methods 17, 391-394 (2020) and McGee, J. P. Anal. Chem. 93, 2723-2727 (2021), each of which are incorporated by reference, for any purpose, herein. In general, the I2MS approach includes five steps. In step 1 of the I2MS approach, hundreds of ions may be observed per acquisition in a random-style trapping event. In step 2, the frequency of each ion signal is determined and analyzed independently. At this processing stage, precise information for each ion, including frequency, intensity and m / z value, is established. Step 3 determines the ion’s signal strength using a data-plotting and data-analysis process that assesses the current induced by an ion on the detection electrodes as a function of acquisition time. This signal strength determination is called the selective temporal overview of resonant ions (STORI) process, with the slope of a STORI plot being proportional to the charge of the ion. In step 4, the charge of the ion is determined by a slope-to-charge calibration function. The STORI slope of an ion with an unknown charge is assigned the closest integer charge state on the calibration function. Finally, in Step 5, using the integer charge (z) and m / z, it is possible to determine the mass of each ion and produce a spectrum in the true mass domain with different spectral properties and increased resolution via centroiding and binning individual ion signals.

[0043] The methods disclosed herein include determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers. Mass heterogeneity may be determined by measuring the centroid from half-maximum of each major, resolved mass-domain peak as a function of the sequential occurrence of each peak, determining the space between each sequential peak, and comparing each of the spaces. A major, resolved peak may be identified by manual selected, for example by their prominences and / or resolution from other peaks, or automated selection by an peak-picking algorithm. For example, a mass scatterplot of resolved peak centroids versus sequential peak number (FIG 2a and FIG 4a) may show a nonlinear trend where mass peaks are periodic, but their period is not constant (i.e., as sequential peak number increases, the masses associated with each peak rises nonlinearly). A nonlinear relationship between mass and peak sequence may indicate, for example, suggest reduced structural uniformity in the oligomer.

[0044] The methods disclosed herein also include determining mass abundance (i.e., mass frequency) of each oligomer. This may be accomplished applying Fourier transforms to the mass spectra. In some cases, subregions of the mass spectra may be analyzed by Fourier transform. In some embodiments, the Fourier transform is a sliding window Fourier transform (z.e., short-time Fourier transform, windowed Fourier analysis). Subregions of the mass spectrum may be chosen for Fourier transform analysis. The subregions may be selected based upon knowledge of masses corresponding to the monomer, dimer, trimer, tetramer, pentamer, hexamer, etc up to the highest number of repeating monomers expected for a given oligomer. For example, the dodecamer having a mass of 55.2 kDa, is the highest order oligomer observed for amyloid beta soluble oligomers at a point where, after growing further, either become toxic or nontoxic. Hence, being able to detect at and around an oligomer mass of 55.2 kDa may be important for diagnosing, assessing disease progression, or assessing treatment efficacy. A subregion for the monomer to dodecamer would include about 0 kDa to about 55 kDa. Coherent signal on the 1 / frequency domain denotes consistent peak spacings of that value, corresponding to recurring building blocks of an oligomer (e.g., amyloid beta monomer, dimer, or trimer). A lack of coherent signal at a given l / frequency domain indicates lack of repeating structure and suggests unstructured aggregates.

[0045] Referring to Figure 13, the methods disclosed herein may be used to diagnose a subject suspected of needing a treatment for an oligomer-associated disease or condition 1008, the method including providing a sample suspected of having one or more oligomers from a subject in need 998, producing, with an ion source, ions of the sample, each of the ions having a mass-to-charge ( / ?? z) ratio 1018, detecting a multiplicity of ions generated with a current detector 1028, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 1038, generating a mass-domain spectrum from the ion masses with the mass- analyzer, the mass-domain spectrum having one or more mass-domain peaks 1048, determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 1058, comparing the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers to one or more diagnostic reference metrics of an oligomer-associated condition 1068. Methods for determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers comprises may comprise identifying mass frequencies by applying one or more Fourier transforms to the mass spectra. In some cases, applying a Fourier transform may include applying a sliding window Fourier transform or a Fourier Transform to a sub-region of interest in the mass spectrum.

[0046] Reference metrics of an oligomer-associate condition refers to data of the outcome (z.e., distinguishing characteristics of at least one oligomer which may be related, correlated, caused by, or associated with a condition associated with one or more oligomers. In some cases, the condition associated with oligomers may include neurodegenerative diseases. For example, amyloid beta oligomeric proteins may be associated with Alzheimer’s disease and alpha-synuclein oligomers are implicated in Parkinson’s disease.

[0047] Reference metrics are preferably obtained from a sample from a subject known to experience a disease or condition associated with one or more oligomers. The reference metrics may also be an average or a mean obtained from such a group of samples. The reference metrics can be obtained by applying the presently disclosed methods. Alternatively, the reference metrics may be obtained from a sample from a subject known to not experience from a condition associated with one or more oligomers or a subject known to have no predisposition to a condition associated with one or more oligomers. The reference metrics may also be an average or a mean obtained from such a group of samples. Reference metrics may also be a reference calculated on the relative or absolute values associated with one or more oligomers of a representative population of individuals apparently healthy or experiencing from a condition associated with one or more oligomers. In some cases, the reference metrics may also be a reference calculated on the relative or absolute values of averages or medians. The population of subjects referred to above includes a plurality of subjects, at least 5, at least 10, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000 subjects. The subject diagnosed by the methods described herein and the subject of said plurality of subjects are of the same species. In one embodiment, the reference metrics may be obtained by calculating a reference metric from an appropriate statistical measure, including mean, median, quantile, logistical regression analysis, or other methods that give a threshold. The reference metric may be stored in a suitable data storage medium, such as a database, and may be used for future diagnosis.

[0048] Referring to Figure 14, the methods disclosed herein may be used to assess treatment of the subject having a disease or condition associated with oligomers 1000. To evaluate the efficacy of a treatment in a subject suspected of having a disease or condition associated with oligomers includes providing a pre-treatment sample having oligomers from the subject in need 992, producing ions of the sample with an ion source where each of the ions having a mass-to-charge (m / z) ratio 1012, detecting a multiplicity of ions generated with a current detector 1022, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 1032, generating a mass-domain spectrum from the ion masses with the massanalyzer 1042, the mass-domain spectrum having one or more mass-domain peaks, and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 1052, administering one or more treatments to the subject in need 1062, providing a post-treatment sample having oligomers from the subject in need 993, producing ions of the sample with an ion source where each of the ions having a mass-to-charge (m z ratio 1013, detecting a multiplicity of ions generated with a current detector 1023, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 1033, generating a mass-domain spectrum from the ion masses with the mass-analyzer 1043, the mass-domain spectrum having one or more mass-domain peaks, and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 1053, and comparing pre-treatment sample and post-treatment sample mass heterogeneity and / or oligomer abundance metrics 1054.

[0049] Referring to FIG. 15, the methods disclosed herein may be used to assess progression of an oligomer-associated disease or condition in a subject 2000. To evaluate the progression a disease or condition associated with oligomers includes providing a first sample having oligomers from the subject in need 1992, producing ions of the sample with an ion source where each of the ions having a mass-to-charge (m z) ratio 2012, detecting a multiplicity of ions generated with a current detector 2022, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 2032, generating a mass-domain spectrum from the ion masses with the mass-analyzer 2042, the mass-domain spectrum having one or more mass-domain peaks, and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 2052, providing a second sample obtained after the first sample is obtained from the subject having oligomers from the subject 1993, producing ions of the sample with an ion source where each of the ions having a mass-to-charge (m z) ratio 2013, detecting a multiplicity of ions generated with a current detector 2023, determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer 2033, generating a mass-domain spectrum from the ion masses with the mass-analyzer 2043, the mass-domain spectrum having one or more mass-domain peaks, and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers 2053, and comparing the first sample and the second sample mass heterogeneity and / or oligomer abundance metrics 2054.

[0050] The mass heterogeneity and / or mass abundance determined in the oligomers of the pretreatment sample, the mass heterogeneity and / or mass abundance determined in the oligomers of the post-treatment sample, and the change between the pre-treatment and post-treatment metrics may indicate a diagnosis or change in diagnosis, a treatment or change in response to a treatment, or a subgroup or change in a subgroup of the subject.

[0051] As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0052] Miscellaneous

[0053] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0054] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0055] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0056] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0057] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0058] EXAMPLES

[0059] Amyloid oligomers are a blind spot in proteomics. Their noncovalent complexation renders analysis by traditional bottom-up or denatured top-down mass spectrometry incomplete, and their immense heterogeneity largely bars informative intact analysis by native top-down mass spectrometry. In this work, Inventors leverage the charge detection technique Individual Ion Mass Spectrometry to maximize spectral clarity. In comparing synthetic amyloid beta oligomers with those derived from the 5xFAD mouse model and immunopurified with the antibodies 4G8 and NU2, Inventors establish peak spacing analysis as an additional tool to thoughtfully scrutinize oligomer populations across both sample type and mass.

[0060] Charge detection MS (CDMS) offers a means of spectral decongestion by measuring ion charge and placing analytes directly in the mass domain instead of the m'z domain. CDMS has been used to measure the composition of synthetic amyloid fibrils (including Ap fibrils), but these studies utilized low-resolution ion trap mass spectrometers and did not provide any information on oligomeric intermediates. In recent years, Orbitrap mass spectrometers have proven capable of high-resolution CDMS. Unlike ion traps, Orbitraps have a high capacity for CDMS multiplexing — 0(100) to 0(1000) as opposed to 0(1) to 0(10) — so long as each analyte signal is individually resolved in m z during each singular detection period. Furthermore “Individual Ion Mass Spectrometry” (I2MS), a specific subsect of Orbitrap CDMS, has a high tolerance for premature ion decay during the detection period. This higher tolerance further increases analytical throughput for small oligomers (<100 kDa). Therefore, combining high resolution and high throughput, I2MS stands uniquely poised to tackle the immense heterogeneity of oligomeric structures. In this work, Inventors leverage the analytical power of I2MS to analyze amyloid beta oligomers (“ApOs”) from both immunopurified 5xFAD mouse samples and synthetic environments. Therefore, this study showcases not only the first light on more granular oligomer spectral features but also the first light on biologically derived samples.

[0061] Materials and Methods

[0062] Mouse Model Preparation.

[0063] Analysis on 15-month-old wild type (WT, for secondary validation) and 5xFAD transgenic mice (Mus musculus) was performed under institutional animal car and use protocols Mice were euthanized by cervical dislocation, and whole mouse brain tissue was harvested and flash frozen prior to extract preparation.

[0064] Tissue Homogenization. The homogenization buffer con-sisted of phenol red-free Ham's F-12 (Thermo Fisher) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher) in a ratio of 1 mL of buffer per 100 mg of brain tissue homogenized. The brain tissue was weighed before being homogenized on ice-cold buffer for 3 cycles of 10s using a handheld homogenizer. The homogenate was then centri-fuged at 14,000 * g for 20 minutes at 4°C. The supernatant was collected in a protein low-bind microcentrifuge tube. The pellet was resuspended in 1% Triton X- 100 in the homogenization buffer, and the supernatant was collected again in a protein low-bind microcentrifuge tube. Protein concentration was determined by Qubit fluorometer according to manufacturer instructions. All collected supernatant was aliquoted into low-binding tubes at 500 pg total protein per tube, frozen using liquid nitrogen, and stored at -80°C. Apo content was validated by sandwich ELISA using oligomer-selective antibodies polyclonal m9328 and NUscl48,49 (Figure 9a), and coarse size distributions of oligomers was determined through the use of molecular weight cutoff (MWCO) spin filters.

[0065] Immunopurification of endogenous oligomers. 25 pg of either 4G8 or NU2 antibody was conjugated to 5 mg Dynabeads M-270 epoxy resin (Thermo Fischer) using the manufacturer’s protocol in 500 pL 1 M ammonium sulfate with 0.1 M sodium phosphate buffer at pH 7.4 (overnight at 37°C with end-to-end rotation). All tubes used in this protocol are low protein binding tubes. Then, 50 pL of conjugated beads were blocked with 0.1% bovine serum albumin with end- to-end rotation at 4°C for 1 hour. Then, the albumin was discarded, and the beads were washed with tris-buffered saline (TBS). 500 pg of total protein brain (cortex and hippocampus) homogenate (diluted in Ham's F-12+protease inhibitor cocktail) was incubated with the beads for 1 hour at 4°C. After binding, the system was washed 3 times with 700 uL 0.1% TBS-Tween 0.1% and 3 times with TBS (changing the carrying tube after the second TBS wash). Then, 30 pL of 150 mM ammonium hydroxide in water was used to elute the oligomers from the beads for 15 min at 4°C under rotation. The eluate was aliquoted, frozen using liquid nitrogen, and stored at -80°C. Purification of A Os from 5xFAD over WT samples was validated using m93 / NUscl ELISA and dot blot probed with anti-ApOs m93 (Figure 10), and reproducibility between different specimens of the same strain was validated using NUscl for an ELISA (Figure 11).

[0066] Creation of synthetic oligomers. Synthetic ApOs were prepared following a procedure established previously. [Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Api-42 are potent central nervous system neurotoxins. Proceedings of the National Academy of Sciences 1998, 95, 6448-6453.] Shortly, AP(l-42) peptide (Echelon Biosciences) film was brought to room temperature and dissolved to 5 mM using di-methyl sulfoxide. Ham’s F12 medium was used to dilute the peptide to 100 pM. The mixture was then vortexed for ~10 seconds and incubated overnight at 4 °C. The next day, the mixture was centrifuged at 14,000 x g for 10 minutes at 4 °C. The supernatant (oligomeric product) was then aliquoted for single-time use and stored at -80 °C. Sample Injection. Samples were diluted l OO-lOOOx in 30 mM ammonium hydroxide in water. Samples were infused at 1-2 pL / min using a heated electrospray (HESI) source at 2.3-2.9 kV and a syringe pump.

[0067] MS Instrumentation. Experiments employed a Q Exactive Ultra High Mass Range (UHMR) Orbitrap (Thermo Fisher Scientific). Data were recorded using I2MS as described previously with a resolution of 140k at 200 m / z (~1 second transient).

[0068] ELISA. Wells were coated with 80 pL of polyclonal a-ApOs m93i (at 2 pg / mL diluted in TBS) for 16-20h at 4°C. The next day, the wells were washed 3x with 250 pL of TBS and blocked with 200 pL Thermo blocking solution (Thermo Fisher) at room temperature for 2 h. After one wash with 300 pL TBS, 80 pL of sample added and incubated for 16-20h at 4°C under mild agitation. The next day, the wells were 3x washed with 300 pL TBS + Tween 20 (Sigma Aldrich) 0.1% (TBS-T) and incubated with 80 pL of phage bound NUscl diluted to an estimated titer of 1.5 x lOn pfu / mL in blocking solution for 2 h at room temperature and mild agitation. Wells were then washed three times with 300 pl of TBS-T and incubated with 80 pL a-M13 HRP-conjugated antibody (Sinobiological) diluted at 0.2 pg / mL in blocking solution for 1 h at room temperature and mild agitation. Finally, wells were washed four times with 300 pL TBS-T, once with TBS, and incubated with 75 pL of TMB substrate (Sigma) for 2-10 minutes in the dark. The reaction was stopped with 40 pL of 0.5 M H2SO4 (Merck). Absorbance was read in a plate reader at 450nm.

[0069] Dot Blot. Synthetic A Os were used as standards, and eluates from immunoprecipitation with NU2 beads from WT and 5xFAD mouse brain extracts were analyzed. 2 pL of each sample (either standards or eluates) was applied onto a nitrocellulose membrane (0.45 pm, GE Healthcare Life Sciences). Membranes were allowed to air dry at room temperature for 15 min, blocked with 3% NFD milk in TBS-T for 2 h at room temperature, and incubated with polyclonal anti-ApOs m93 at 2 pg / mL in blocking solution for 16-20h at 4°C. After 3, 5-minute washes in TBS-T, membranes were incubated with HRP-conjugated a-rabbit IgG secondary antibody (GE Healthcare Life Sciences) at 0.2 pg / mL in blocking solution for 1 h at room temperature. After 3, 5-minute washes in TBS-T and a final wash in TBS, membranes were exposed to SuperSignal West Atto chemiluminescent detection substrate (Thermo Fisher) and imaged on a SapphireTM system (Azure Biosystems).

[0070] Cryogenic Transmission Electron Microscopy. Copper grids with 300 mesh and a carbon film (EMS. CF300-Q1-50) were glow discharged in a Pelco easiGlow glow discharger (Ted Pella, 91040) for 20 seconds at 0.26 mBar in a 15 mA atmosphere plasma in order to allow sample to spread evenly on the otherwise hydrophilic carbon surface. Thirty seconds after drop-casting 5 pL of sample on the grid, the droplet was wicked away with filter paper, but the grid was not allowed to dry prior to applying 5 pL of 1% Uranyl Acetate (mixed from stock powder; SPI, 02624-AB) as a negative stain. The negative stain droplet was wicked up and again replaced three times without tire grid being allowed to dry. Hie final droplet of negative stain sat in place on the grid for four minutes before wicking it off with filter paper; the grid was given >4 minutes to dry before being loaded into a Hitachi HD2300 cFEG Scanning Transmission Electron Microscope for viewing. Image data was gathered with a Digiscan system within Gatan Digital Micrograph software while utilizing the high angle annular dark field and transmission phase contrast detectors within the microscope.

[0071] Results and discussion

[0072] By utilizing I2MS, Inventors were able to place ApOs directly into the mass domain (Figure 1, Figure 3). Note that all samples were taken in technical duplicate (i.e., two aliquots from the same biological replicate), but a single replicate is shown in Figures 1 and 2. Replicates in all modes of analysis used in the main text are shown in the Supporting Information (Figure 3, Figure 4) and show general concordance across replicates. I2MS allows the user to acquire conventional m z data (Figure la) concurrently with the data output used to create a mass spectrum in 300 Da bins (Figure lb). The m,z spectra exhibit qualitative differences that cannot be deconvolved due to the lack of a resolved charge state distribution. The mass domain spectra exhibit broad but periodic peaks that have distinct signatures from one sample type to another (Figure lb, enlarged region). The latter of these data types may be expressed as heat maps in terms of assigned charge (Figure 1c).

[0073] The broad width of each peak bars precise intact mass annotation of these structures. However, it is still possible to analyze these peaks within their greater spectral context (Figure 2, Figure 4, Table 1). Specifically, Inventors plotted the centroid of each major, resolved peak (centroid from half-maximum) as a function of their place in their respective peak series (Figure 2a). Then, Inventors subjected subregions of the mass spectra from Figure lb to Fourier transforms in order to identify component mass frequencies (i.e., the most consistent oligomeric building blocks) (Figure 2b). Spectra were sampled 12-55 kDa and 55-165 kDa, using the approximate size of a dodecamer to mark the boundary between the sampled regions. Table 1 . Tabulated peak centroids used in Figure 2a and Figure 4a. The immense heterogeneity depicted in the conventional mass spectrum (Figure la) highlights the immense barrier the community faces in interpreting distributions of amyloid beta oligomers. In general, proteins and protein complexes analyzed by native MS exhibit a correlation between mass and m / z. To this end, the synthetic and NU2-based (“NU2”) samples appear to share some features: a primary distribution ranging 2000-7000 m'z and a secondary distribution ranging 8000-9000 m / z. The 4G8-based sample (“4G8”) exhibits differing features from the other NU2 and synthetic samples: a primary distribution below 2000-5000 m z and a secondary distribution ranging 8000-12000 m / z. Overall, the m / z results are unusual and difficult to interpret. The I2MS data type is collected in acquisitions where each ion is individually resolved in m / z space, and each acquisition typically exhibits an “individual ion shelf’ that increases in signal intensity with decreasing m / z as explored in integrated-signal-based Orbitrap charge detection. However, A^Os exhibited a “reverse” individual ion shelf; signal intensity increased with increasing m / z (Figure 5). This trend suggests that the mass heterogeneity of the A0Os is broad and “outpaces” the charge heterogeneity typical of electrospray ionization. NU2 was of particular interest for secondary validation due to the antibody targeting oligomers and being applied to a mouse model, so Inventors imaged NU2 oligomers with cryogenic transition electron microscopy (Figure 6). The spherical shape of the oligomers observed as well as their average size (11 ±2 nm in diameter, n = 29) is consistent with prior AFM data on synthetic oligomers. Inventors also observed oligomer seeding off of a fibril, mirroring the findings of Economou et al. In other words, the imaging data of the NU2 data, in combination with ELISA and dot blot data (Figure 9-11), supports the presence of oligomers despite the unusual nature of the m z data compared to prior ventures.

[0074] The mass domain data adds immense clarity as to the presumptive similarities and differences noted from the m / z data (Figure lb). Specifically, while both NU2 and synthetic exhibit two distributions coarsely divided by the approximate mass of a dodecamer of canonical A0(l-42) (-55 kDa), the precise peak spacings are distinct from one another (Figure lb, enlarged region). The presence of a wide distribution of oligomer masses is sup-ported via ELISA analysis on the retentate and filtrate of 30 kDa and 100 kDa MWCO spin filtering (Figure 9b). The low-mass series in 4G8 appears more tightly spaced, and the secondary, high-mass distribution does not exhibit any sort of resolved, orderly peak series. The resolved peaks in 4G8 diminish well before the dodecamer mass. Despite the massive heterogeneity of not only each resolved peak but also each mass spectrum, each data set was collected in about an hour (3600 acquisitions, averaging -200-500 detected ions per acquisition), demonstrating the massive overall throughput of the method compared to traditional charge detection techniques (Figure 7).

[0075] Reframing the charge-assigned data as heat maps strongly highlights how the mass heterogeneity of the oligomers outpaces their charge heterogeneity (Figure 1c). In all three samples, the primary distribution seems extraordinarily charge-constrained, typically only having one charge state at any given mass. This lies in stark contrast to heat maps of protein standards that are strongly constrained in mass while simultaneously exhibiting charge heterogeneity (Figure 8). The trends observed in the heat maps support the “reverse individual ion shelves” seen in Figure 5; higher mass near-uniformly correlates with higher charge for the oligomer data sets. This strict charge constraint indicates a structural constraint not typically seen in globular proteins, as structure in globular proteins dictate surface accessibility of residues that may carry charge in electrospray ionization (which imparts a stochastic amount of charge). While an unexpected result, the trend is consistent with structural data on amyloid beta structure’s that suggests that few of the monomer’s charge-carrying residues are exposed to the bulk solution in fibril form. High-mass 4G8 oligomers deviate from charge constraint by showing much greater charge heterogeneity (Figure 1c, left). This observation coupled with the loss of resolved peak patterns at high mass despite sizeable signal indicates that the high-mass structures observed in 4G8 are not nearly as charge-constrained — nor, consequently, structure-constrained — as elsewhere throughout the data series. This is consistent with the fact that the 4G8 antibody targets a monomeric epitope of amyloid beta. Therefore, 4G8 should be capable of pulling down both lower-mass structures where the epitope may be exposed and high-mass aggregates where there is no unified structure.

[0076] The structural constraint for NU2 and synthetic samples does not equate to structural uniformity. The mass scatterplot of resolved peak centroids (Figure 2a) clearly shows that the rate of mass increase from peak to peak grows throughout the NU2 and synthetic peak series (4G8 does not exhibit a sufficiently extensive peak series to observe the same trend). In other words, the mass peaks are clearly periodic as per Figure lb, but their period is not constant. The sliding window Fourier transforms highlight this trend (Figure 2b). 4G8 exhibits a sharp frequency peak corresponding to the mass of monomeric amyloid beta. While the synthetic sample also contains a small monomeric frequency below 55 kDa, the dominant signal for both synthetic and NU2 below 55 kDa corresponds to a dimer. In other words, below a dodecamer, 4G8 peaks are distinctly separated by monomeric masses, whereas NU2 and synthetic peaks are distinctly separated by dimeric masses. This finding suggests that the NU2 and synthetic oligomers at low masses are structurally constrained such that amyloid beta is not recruited in monomeric quantities.

[0077] The Fourier transforms for masses 55-165 kDa further highlight the features discussed above. 4G8 has no dominating frequency in this range compared to the baseline, which supports the above assertion that its high-mass components are unstructured aggregates. The dominant frequencies of NU2 and synthetic both shift towards the trimer stroichiometry. The literature does support the trend of oligomers utilizing larger and larger building blocks during the early stages of amyloid beta fibril formation. While trimers are not mentioned in this pathway for A0(1 -42), there is evidence to suggest that trimeric amyloid beta is stable and does not undergo fibrilization. The significance of these detected spacings in the Fourier transforms and how they change across the mass domain would have to first be validated through a comparative study of controlled oligomeric populations where the projected toxicity of each population is known.

[0078] Conclusions

[0079] Inventors were able to probe endogenous ApOs in novel ways establish impactful modes of comparing distributions while pushing the boundaries in what type of samples can be analyzed, namely endogenous oligomers. Most notably, the differences in oligomeric distributions can be traced through not only mass differences but also spacing differences, all of which point towards established principles in the underlying biology. Through this foundation, there is strong motivation to pursue follow-up studies. For example, establishing a consistent correlation between oligomeric population toxicity and the presence of dimers or trimers in the inverse frequency space would provide immense value to the medical community in understanding whether potential therapeutics reduce toxic species of oligomers. Furthermore, this MS technique is not specific for ApOs. Therefore, I2MS-based techniques stand poised to positively impact oligomer studies far beyond AD.

Claims

CLAIMS1. A method for oligomer characterization, the method comprising: providing a sample having one or more oligomers; producing, with an ion source, ions of the sample, each of the ions having a mass- to-charge (m z) ratio; detecting a multiplicity of ions generated with a current detector; determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer; generating a mass-domain spectrum from the ion masses with the mass-analyzer, the mass-domain spectrum having one or more mass-domain peaks; and determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers.

2. The method of claim 1, wherein determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers comprises: determining mass heterogeneity by determining the centroid of each mass-domain peak as a function of the sequential occurrence of each peak; determining the space between each sequential peak; and comparing each of the spaces.

3. The method of claim 1, wherein determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers comprises identifying mass frequencies by applying one or more Fourier transforms to the mass spectra.

4. The method of claim 3, wherein the one or more Fourier transforms is a sliding window Fourier transform.

5. The method of claim 3, wherein the one or more Fourier transforms are applied to subregions of interest of the mass spectrum.

6. The method of claim 1, further comprising at least partially isolating the oligomers from a sample.

7. The method of claim 6, wherein the at least partial isolation is accomplished by immunopurification, affinity purification, membrane-based filtering, chromatography, electrophoresis, or any combinations thereof.

8. The method of claim 7, wherein the immunopurification comprises antibody-conjugated beads.

9. The method of claim 1, wherein the sample comprises one or more oligomers that originate from an in vivo source, one or more oligomers that originate from an in vitro source, one or more oligomers that originate from an ex vivo source, one or more synthetic oligomers, or any combination thereof.

10. A method of diagnosing a subject suspected of needing a treatment for an oligomer- associated disease or condition, the method comprising: providing a sample suspected of having one or more oligomers from the subject; producing, with an ion source, ions of the sample, each of the ions having a mass-to-charge (m 'z) ratio; detecting a multiplicity of ions generated with a current detector; determining ion masses for each of the multiplicity of ions detected with the current detector with a mass analyzer; generating a mass-domain spectrum from the ion masses with the mass-analyzer, the mass-domain spectrum having one or more mass-domain peaks; determining one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers; and comparing the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers to one or more diagnostic reference metrics of an oligomer-associated condition.

11. The method of claim 10, wherein the oligomer-associated disease or condition is a neurodegenerative disease.

12. The method of claim 10, wherein the oligomer-associated disease or condition is Alzheimer’s disease or Parkinson’s disease.

13. The method of any one of claims 10-12 further comprising administering the subject a treatment for the oligomer-associated disease or condition if the one or more metrics capturing mass heterogeneity and / or mass abundance of oligomer are indicative for the oligomer-associated disease or condition.

14. The method of claim 13 further comprising assessing efficacy of treatment.

15. The method of any one of claims 10-12 further comprising assessing progression of the oligomer-associated disease or condition if the one or more metrics capturing mass heterogeneity and / or mass abundance of oligomer are indicative for the oligomer- associated disease or condition.

16. A method of assessing efficacy of treatment of a subject having an oligomer-associated disease or condition, the method comprising: providing a pre-treatment sample containing one or more oligomers from the subject before treatment is administered; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the pre-treatment sample according to the method of any one of claims 1-9; administering one or more treatments to the subject; providing a post-treatment sample containing one or more oligomers from the subject after the one or more treatments are administered; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the post-treatment sample according to the method of any one of claims 1-9; andcomparing the one or more metrics capturing the heterogeneity and / or abundance of oligomers in the pre-treatment sample to the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers in the post-treatment sample.

17. The method of claim 16, wherein the oligomer-associated disease or condition is a neurodegenerative disease.

18. The method of claim 16, wherein the oligomer-associated disease or condition is Alzheimer’s disease or Parkinson’s disease.

19. A method of assessing progression of an oligomer-associated disease or condition in a subject, the method comprising: providing a first sample containing one or more oligomers from the subject; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the first sample according to the method of any one of claims 1-9; providing a second sample obtained after the first sample is obtained from the subject containing one or more oligomers; determining one or more metrics capturing the heterogeneity and / or abundance of oligomers in the second sample according to the method of any one of claims 1-9; and comparing the one or more metrics capturing the heterogeneity and / or abundance of oligomers in the first sample to the one or more metrics capturing the mass heterogeneity and / or mass abundance of oligomers in the second sample.

20. The method of claim 19, wherein the oligomer-associated disease or condition is a neurodegenerative disease.

21. The method of claim 19, wherein the oligomer-associated disease or condition is Alzheimer’s disease or Parkinson’s disease.

Images