# Patent

## Title
Method, composition, isolation and identification of a plaque particle and related biomarker

## Abstract

The disclosure relates to an in vitro technology of detecting presence of a plaque particle, isolating the plaque particle followed by its composition analysis in several diseases states or before the disease sets in. A mechanism and a process leading to plaque formation, identifying a component in the mechanism of plaque formation, an identification of a biomarker for diagnosis/early diagnosis of plaque associated disease is described. A method of screening a candidate agent as an anti-plaque agent using flow cytometer, mass spectrometer and specific biomarkers is performed. Provided also are kits for use in practicing embodiment of the methods. A plaqueproteome database is also generated with novel protein sequences for diagnosis and specific antibodies for specific proteins are also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from and is a continuation-in-part (CIP) of PCT/US2012/066412 filed on Nov. 21, 2012 which is incorporated herein by reference.


FIELD OF TECHNOLOGY
The present application relates generally to methods and compositions relating to formation of multiple types of plaque particles in biological samples and their detection, isolation and biomarkers identification. More specifically, the present application relates to use of Flow cytometer and Mass spectroscopy for isolation and identification of molecules that contribute to formation of plaque particles.
BACKGROUND
Plaque development is a complex pathogenic process occurring mainly due to abnormal deposition of molecules such as cholesterol, lipids, amyloid peptides, metals and metabolites in blood vessels (Viola M et al, 2013; Thal D R et al, 2008). These molecules, while existing in the soluble form, are involved in the normal functions of key biochemical pathways, however, their abnormal metabolism is implicated to origin of many vascular and neurodegenerative disorders (Wirth M et al, 2013; Brown W R, 2011). More specifically, transformation of cholesterol and amyloid peptides from their initial soluble form into oligomers or aggregates and finally into insoluble plaque particles is the primary cause in the development of atherosclerotic and amyloid plaques.
The plaque forming aggregates once formed inappropriately interact with a wide range of bio-molecules present in the surrounding extracellular or intracellular spaces. Endogenous physiological pathways such as proteolytic and immune system play critical role in the clearance of the aggregates. However, under abnormal conditions defective clearance of the plaque aggregates or oligomers leads to their progressive accumulation in the coronary and cerebrovascular regions of blood vessels ultimately leading to fatal symptomatic events such as Myocardial Infarction and dementia (Sultan M, 2014; Grimm Mo. et al. 2013).
Although, the major components present in the atherosclerotic and amyloid plaques have been identified as the underlying biochemical mechanism of plaque assembly process, however it is not completely understood. This is mainly due to the fact that in vivo plaque development is a slow pathological process covering a larger window period of time spanning years to decades since the early asymptomatic stage to occurrence of symptomatic events.
Currently, autopsy and biopsy samples are used to examine plaque composition with limited success and often these samples are contaminated with host cell and tissue contributing to misleading scientific observations leading to a gap in diagnosis and treatment.
SUMMARY
The present disclosure relates to an in vitro technology of detecting presence of a plaque particle, isolating the plaque particle followed by its composition analysis. Further, the present application also relate to understanding a mechanism of or a process leading to plaque formation, identifying a component in the mechanism of plaque formation and a method of screening a candidate agent as an anti-plaque agent. The present disclosure also relates to identifying a bio-marker of plaque formation leading to early diagnosis or diagnosis of plaque associated disease. More specifically, the present disclosure relates to an in vitro technology of isolating at least one plaque particle or plurality of plaque particles using a flow cytometer, separating the plaque particle based on different physical parameters; analyzing the particle through Mass Spectroscopy and analyzing a bio-molecule or plurality of bio-molecules present in the plaque particle followed by identifying a bio-marker. Also provided are kits for use in practicing embodiments of the methods.
In one embodiment, the present application relates to a method, comprising: preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or a plaque oligomer is linked to a detectable signal; contacting a biological sample from a subject with the plaque aggregate or the plaque oligomer to form an insoluble plaque particle; isolating the plaque particle; and analyzing a bio-molecule in the plaque particle to identify composition of plaque particle. In another embodiment, the present application relates to a method, comprising: preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or the plaque oligomer is linked to a detectable signal; contacting a biological sample from a subject with the plaque aggregate or the plaque oligomer to form an insoluble plaque particle, wherein the subject is previously diagnosed with a plaque associated disease; isolating the plaque particle using a flow cytometer; and analyzing a bio-molecule in the plaque particle using mass spectroscopy to identify composition of plaque particle.
In one embodiment, the present application relates to a method, comprising: preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or the plaque oligomer is linked to a detectable signal; contacting a biological sample from a subject with the plaque aggregate or the plaque oligomer; isolating a plurality of plaque particles; and analyzing a plurality of bio-molecules in the plaque particles to identify a biomarker or a plurality of biomarkers in the biological sample. In some embodiments, the contacting is with at least one plaque aggregate or at least one plaque oligomer whereas in another embodiment, the contacting is with a plurality of plaque aggregates or a plurality of plaque oligomers.
In most embodiments, analysis of at least one bio-molecule or a plurality of bio-molecules as disclosed may lead to an identification of a biomarker for early diagnosis of plaque associated disease.
In one embodiment, a biological sample is a biological fluid wherein the biological fluid is selected from the group consisting of blood, plasma, serum, cerebral spinal fluid, urine and saliva. In another embodiment, a biological fluid may be a clinical or a non-clinical fluid sample.
In one embodiment, the contacting of biological sample is with at least one plaque aggregate, oligomer or a self-formed plaque particle wherein the contacting of biological sample with at-least one plaque aggregate, oligomer or a self-formed plaque particle triggers a mechanism leading to a formation of at-least one plaque particle. The plaque particle as formed in vitro resembles a plaque associated with Atherosclerosis, Alzheimer's disease (AD), Autism, Parkinson's disease (PD), Multiple Sclerosis (MS), Osteoarthritis, Mad Cow Sponsiform (MCS), Type II diabetes, Dementia, Systemic Amyloidosis (SA), Dialysis-related Amyloidosis, Huntington disease, Levy bodies, Lysozyme myloidosis, Insulin-related Amyloidosis, Amyotrophic Lateral Sclerosis (ALS) and/or other plaque-related and associated disorders.
The subject as disclosed in the present application may be a person or a patient diagnosed previously with a plaque associated disease which may include but not limited to Atherosclerosis, AD, Autism, PD, MS, Osteoarthritis, MCS, Type II diabetes, Dementia, SA, Dialysis-related Amyloidosis, Lysozyme Amyloidosis, Huntington disease, Levy bodies insulin-related Amyloidosis, ALS and/or other plaque-related and associated disorders. In some embodiments, the subject may be a person or a patient at risk of having, suspected of having or a family history of having plaque associated disease. In some embodiments, the method further comprises diagnosing or stratifying subjects based on plaque particle formation, plaque particle sub-types, plaque particle images, plaque particle count, or plaque particle profile.
In some embodiments, the detecting label may be a fluorescent, chemiluminscent, radio-labeled, enzymatic or an antibody labeled with fluorescence or metal and thus may be detected using known assays. In a preferred embodiment, the detectable label may be a fluorescent label, thus detecting at least one fluorescent labeled plaque aggregate, plurality of plaque aggregates or a pair of plaque aggregates labeled with different fluorophores using fluorescence resonance energy transfer (FRET).
In one embodiment, at least one plaque aggregate, plaque oligomer or self-formed plaque particle comprises one or more of the following: protein, protein derivative, cholesterol, cholesterol derivative, lipid, lipid derivative, Abeta-42, Abeta derivatives, Synuclein, prion, Amylin, Tau, Tau derivative, phospholipids, cholesterol crystals, Serum Amyloid A, Beta Microglobulin, lysozyme, insulin, or super dioxide dismutase, and calcium-phosphate (CP).
In one embodiment, the present disclosure further relates to a method of screening a candidate agent as an anti-plaque agent. The candidate agent may act to prevent, inhibit or stimulate the in-vitro formation of the plaque particle. The method of screening a candidate agent as disclosed, comprises: preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or the plaque oligomer is linked to a detectable signal; contacting a biological sample from a subject with the plaque aggregate or the plaque oligomer; adding the candidate agent or libraries of candidate agents; isolating the plaque particle; and analyzing a bio-molecule in the plaque particle and comparing it with a bio-molecule identified in the plaque particle isolated from biological sample not in contact with the candidate agent to screen the candidate agent as an anti-plaque agent. The candidate agent may be added before the contacting or after the contacting with the biological sample. In most embodiments, the biological sample where the candidate agent is not added before and after the contacting is considered as a control sample for screening methods. A candidate agent as disclosed, may include but not limited to a chemical compound, a small molecule, a therapeutic drug, a biological molecule, a natural compound, a natural or a synthetic oligomer, a ligand, a protein, an antibody and/or other component capable of binding the plaque aggregate, plaque oligomer, self-formed plaque particle or plaque particle in the presence or absence of biological sample, preventing their assembly, disassembling these aggregates, oligomers, self-formed plaque particles or plaque particles once already formed, or reducing their pathogenic properties. The screening method will further identify candidate agent for their potential as therapeutics for diagnosing, preventing, treating, and/or curing plaque related diseases.
Thus, in one embodiment, the disclosure relates to a method of screening a candidate agent comprising: preparing at least one plaque aggregate or a plaque oligomer in vitro wherein the at least one plaque aggregate or plaque oligomer is linked to a detectable label; culturing mammalian cells with the at least one plaque aggregate or plaque oligomer wherein the mammalian cells express morphologic changes, pathological symptoms, cell adhesion molecules, cytokines and or apoptosis, inflammation; contacting the mammalian cells at least one candidate agent; and then identifying candidate agent that prevent or lessen the formation of pathological symptoms or morphological changes in the cells.
In one embodiment, a mechanism of plaque formation will also be evaluated by disclosed technology. The mechanism may be delineated by the bio-molecule analyzed and quantitated before and after the formation of plaque aggregates. In one embodiment, the present disclosure relates to a method, comprising: preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or the plaque oligomer is linked to a detectable signal; analyzing a bio-molecule in a biological sample from a subject; contacting the biological sample with the plaque aggregate or the plaque oligomer; detecting the plaque particle; isolating the plaque particle; analyzing a bio-molecule in the plaque particle; and comparing the biomolecule analyzed before and after the contacting of the biological sample to delineate a mechanism of plaque formation. In another embodiment, the method as disclosed can further be elaborated to identify a biomarker or plurality of biomarkers for diagnosis and early diagnosis of plaque related disease. The bio-molecules as identified may then be correlated to the biochemical pathways indicating a mechanism of plaque formation.
In one embodiment, a method to prepare a plaque forming oligomer or aggregate is disclosed. In another embodiment, a plaque forming cholesterol, phospholipid and amyloid oligomer or aggregate is disclosed. The plaque forming oligomer or aggregate as synthesized may then be used for in vitro plaque formation as described.
In one embodiment, the present disclosure relates to a method of sorting and isolating a plaque particle synthesized in the biological sample such as a bio-fluid. In one embodiment, the present disclosure relates to a method of sorting and isolating the plaque particle synthesized in the bio-fluid using a flow cytometer. The method as disclosed comprise of preparing a plaque aggregate or a plaque oligomer in vitro, wherein the plaque aggregate or the plaque oligomer is linked to a detectable signal; preparing a plaque aggregate in vitro using at least one of a plaque biomarker, plaque oligomer, plaque antibody and a combination thereof; contacting the biological sample with the plaque aggregate or the plaque oligomer; detecting a formation of the aggregate via the detectable signal; introducing the aggregate to a flow cytometer; performing a dot blot analysis; separating the aggregate particle; and collecting the separated particles for further composition analysis. The aggregate particles may be separated based on differences in density, mass or complex formation.
In one embodiment, the present disclosure relates to a method of processing an isolated plaque particle for its composition analysis which may be a proteomics, lipidomics, glycomics and metabolomics based composition analysis. In most embodiments, the present disclosure relates to a method of processing an isolated plaque particle for mass spectroscopy based proteomics, lipidomics, glycomics and metabolomics analyses.
In another embodiment, the present disclosure relates to a kit for preparation of a plaque aggregate or a plaque oligomer whereas in another embodiment, the present disclosure relates to a kit for segregating and isolating plaque particle. In another embodiment, the present disclosure also relate to a kit comprising: collecting a biological sample from a subject, contacting it to a plaque oligomer; estimating a plaque particle; segregating and isolating the plaque particle and analyzing the biomarker. In most embodiments, the present disclosure relates to a kit for estimating a biomarker for plaque related diseases in a biological sample from a subject which may be a person or a patient at risk of having, suspected of having or a family history of having plaque associated disease. In one embodiment, a plaqueproteome database is compiled. In another embodiment, antibody leads that may be used as a plaque oligomer, plaque aggregate is disclosed.
The method, composition, isolation and identification of a plaque particle and related biomarker disclosed herein may be implemented in any means for achieving various aspects, and may be executed in a form suitable for the mammal and or other vertebrate animals. Other features will be apparent from the accompanying drawings and from the detailed description that follows.



BRIEF DESCRIPTION OF THE DRAWINGS
Example embodiment are illustrated by way of example and no limitation in the accompanying Figures and tables, like references indicate similar elements and in which:
FIG. 1 represents a schematic of the flow cytometer and Mass spectroscopy based detection, sorting and biomarkers identification in plaque particles.
FIGS. 2A, 2B and 2C represents a flow cytometer based detection of Abeta-42 plaque particles.
FIGS. 3A, 3B and 3C represents a flow cytometer based detection of Abeta-28 plaque particles.
FIGS. 4A, 4B and 4C represents a flow cytometer based detection of cholesterol plaque particles.
FIGS. 5A, 5B and 5C represents a flow cytometer based detection of phospholipids plaque particles.
FIGS. 6A, 6B and 6C represents a flow cytometer based sorting and isolation of cholesterol plaque particles.
FIGS. 7A, 7B and 7C represents a flow cytometer based sorting and isolation of high density and low density cholesterol plaque particles.
FIGS. 8A, 8B and 8C represents a flow cytometer based sorting and isolation of Abeta-42 plaque particles.
FIGS. 9A, 9B and 9C represents a flow cytometer based sorting and isolation of Abeta-28 plaque particles.
FIGS. 10A, 10B and 10C represents a flow cytometer based sorting and isolation of Tau (275-305) plaque particles.
FIGS. 11a, 11B and 11C represents a flow cytometer based sorting and isolation of alpha-synuclein plaque particles.
FIGS. 12A, 12B, 12C, 12D and 12E represents a mass spectrum data generated to deduce peptide sequences.
FIGS. 13A, 13B, 13C and 13D represents comparative analysis of common and specific proteins identified in plaque particles.
FIG. 14 represents a pie chart showing clusters of proteins identified for the plaque particles formation and their link to multiple biochemical pathways.



DETAILED DESCRIPTION
Several methods, composition, isolation techniques, kit, and identification procedures of a plaque particle, related biomarkers and plaque specific antibody leads are discussed in the instant disclosure. Although the present embodiment have been described with reference to specific example embodiment, it will be evident that various modifications and changes may be made to these embodiment without departing from the broader spirit and scope of the various embodiment. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
There is currently a lack of in vitro technology to efficiently isolate plaque particles which is hampering efforts to identify biomarkers that could help to better understand the mechanism of plaque formation, lead to early diagnosis and discovering efficient drugs to treat affected patients. Previously, an innovative Plaque Array method for in vitro detection and quantitation of serum derived plaque particles has been developed related to Atherosclerosis and AD (Madasamy, 2011, PCT/US2012/066412). In the present disclosure, a combination of flow cytometer and mass spectroscopy based approach is disclosed for an in vitro isolation, quantitation and biomarker identification from a wide range of plaque particles related to atherosclerosis, amyloidosis and other plaque related diseases.
As used herein, Abeta-42 refers to Amyloid beta peptide 1-42 and derivatives; Abeta-28 refers to Amyloid beta peptide 1-28 and derivatives; Abeta-17 refers to Amyloid beta peptide 1-17 and derivatives; Chl refers to cholesterol; LS refers to phospholipid; and CP refers to calcium phosphate.
The “plaque particles” and “in vitro plaque particles” disclosed herein refer to the same reaction product formed in the presence of added biological sample and the terms are used interchangeably. These terms are different from the term “self-formed plaque particles” which are formed in the absence of added biological sample. “Self-formed plaque particles” refers to one type of reagent used in the plaque array assay.
The plaque aggregates (including but not limited to) such as cholesterol plaque aggregate, phospholipid plaque aggregate, Abeta plaque aggregate, Tau plaque aggregate, alpha-synuclein aggregate, hybrid plaque aggregate and the like disclosed herein are water soluble. The self-formed plaque particle and the plaque particle disclosed herein however are water insoluble. The aggregates of various amyloid peptides disclosed herein as Abeta aggregates generally referred to in the literature as oligomers. As disclosed herein, an array or a panel refer to a plurality of plaque aggregates or self-formed plaque particles.
The plaque aggregates, plaque oligomers or self-formed plaque particles as disclosed to be used in screening biological samples or bio-fluids effect on plaque particle formation may comprise one or more of the following: Abeta peptides and derivatives:







TABLE 1







Shows the human sequences and their representative names.









Sequence




number
Name
Sequence





SEQ ID NO: 1
Abeta 1-42
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG-




VVIA


 


SEQ ID NO: 2
Abeta 1-28
DAEFRHDSGYEVHHQKLVFFAEDVGSNK


 


SEQ ID NO: 3
Abeta 1-17
DAEFRHDSGYEVHHQKL


 


SEQ ID NO: 4
Abeta 22-35 
EDVGSNKGAIIGLM


 


SEQ ID NO: 5
Amyloid 
DAEFRHDSGYEVHHQKLVFFAEDVGCNKG-



(1-42 S26C)
AIIGLMVGGVVIA


 


SEQ ID NO: 6
Amyloid 
E22V-DAEFRHDSGYEVHHQKLVFFAVDVGSNKG-



(1-42)
AIIGLMVGGVVIA


 


SEQ ID NO: 7
Amyloid 
N27A-DAEFRHDSGYEVHHQKLVFFAEDVGSAKG-



(1-42)
AIIGLMVGGVVIA









Additionally sequences of Synuclein, prion, Amylin, Tau, phospholipids, cholesterol crystals, Serum Amyloid A, Beta Microglobulin, lysozyme, insulin, or super dioxide dismutase should also be considered.
In one embodiment, plaque aggregate, plaque oligomer or self-formed plaque particle comprise of at least one component known to be present in in vivo formed plaques in subjects with plaque related disease. In these embodiment, the component of may be linked to a detectable label. In other embodiment, plaque aggregates, plaque oligomer or self-formed plaque particles comprise of cholesterol or its derivatives. In another embodiment, plaque aggregates, plaque oligomer or self-formed plaque particles comprise phospholipid or its derivatives. In one embodiment, the plaque aggregates or the plaque oligomer comprise a single component while in other embodiment they are hybrid aggregates or oligomers and comprise more than one component.
In one embodiment, the plaque aggregates, plaque oligomer and self-formed plaque particles are prepared in phosphate buffered saline (PBS) or phosphate buffers. As an alternate any suitable aqueous solution may be used instead. In another embodiment, the plaque aggregates, plaque oligomer or self-formed plaque particles are prepared using organic solvents such as alcohol. In one embodiment, the reactions forming plaque aggregates and self-formed plaque particles are performed at 37° C. In other embodiment, the reaction is performed at a temperature and a time which are appropriate for progression of a reaction. In one embodiment the reactions using the plaque aggregates, plaque oligomer and self-formed plaque particles in diagnostic or drug discovery or development or other context are performed at 37° C. In other embodiment, the reaction is performed at a temperature and a time which are appropriate for progress of a reaction.
Any biological sample may be tested according to the disclosed methods. Such a sample may be cells, tissue, blood, urine, semen, or a fraction thereof (e.g., plasma, serum, urine supernatant, urine cell pellet or nerve or endothelial cells), which may be obtained from a patient or other source of biological material, e.g., autopsy sample or forensic material. Prior to contacting the plaque aggregates or plaque oligomer, the sample may be processed to isolate or enrich the sample for the desired molecules using a variety of standard laboratory practices which may be used for this purpose, such as, e.g., centrifugation, immuno-capture, cell lysis. Bio-fluid is one category of biological sample. As disclosed herein, the term bio-fluid is a fluid biological sample and is used interchangeably with the term biological fluid. While the bio-fluid used in the examples disclosed herein is serum from human subjects, in one embodiment the bio-fluid may also comprise of plasma, saliva, urine, cerebrospinal fluid and/or blood.
Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples may also include sputum, bronchial washing, bronchial aspirates, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, biological fluids such as cell culture supernatants, tissue, cell, and the like.
The present application discloses an in vitro technology of detecting presence of a plaque particle, isolating the plaque particle followed by its composition analysis. Further, the present application also relate to understanding a mechanism of plaque formation, identifying a component of mechanism of plaque formation and a method of screening a candidate agent as an anti-plaque agent. More specifically, the present disclosure relates to an in vitro technology of isolating at least one plaque particle or plurality of plaque particles using a flow cytometer, separating the plaque particle based on different and selected physical parameters; analyzing the particle through mass spectroscopy and identifying bio-molecules present in the plaque particle to predict a biomarker.
Further, the disclosure relates to a use of a plaque array method to screen candidate agents for the inhibition or stimulation of the in vitro formation of the plaque particles. As such, candidate agents including but not limited to chemical compounds, small molecule compounds, therapeutic drugs, biological molecules, oligomers, ligands, proteins, antibodies or other components, capable of binding the plaque aggregates or self-formed plaque particles or plaque particles in the presence or absence of bio-fluids, preventing their assembly, disassembling these aggregates or self-formed plaque particles or plaque particles once already formed, or reducing their pathogenic properties, are tested for their potential as therapeutic leads for diagnosing, preventing, treating, an d/or curing amyloid or atherosclerosis related plaque diseases. Since the methods or processes disclosed herein are capable of isolating the steps of in vitro plaque particle formation, anti-plaque agents targeting different stages of plaque development are also capable of being identified. The term “anti-plaque agents” and “anti-plaque therapeutics” are used interchangeably herein and refer to compounds or drugs which are effective in: a) dissolving, inhibiting or disrupting the architecture, or structure of a plaque aggregates or self-formed plaque particles or plaque particles described herein; and/or b) inhibiting, preventing, or alleviating the detrimental effects that the plaque may have on other cells, tissues or organs of humans.
In one embodiment, plaque particle(s) as formed following contacting are analyzed through plaque array technology. The plaque array technology permits the discovery of both novel mechanisms and molecules that catalyze the accelerated plaque particle assembly when treated with the biological samples. In one embodiment the plaque array enables the evaluation of the pathogenicity of plaques of varying compositions.
Thus, the present application discloses a methodology of isolating bio-molecule from a plaque particle with the following steps:

    a. Preparation of plaque forming oligomer or aggregates;
    b. Detection of plaque particles;
    c. Sorting and isolation of plaque particles;
    d. Processing of isolated particles for proteomics, glycomics, lipidomics and metabolomics analyses;
    e. Spectroscopic analysis of peptides for identification of different proteins;
    f. Spectroscopic analysis of lipids composition; and
    g. Analyses of the glycans and metabolites composition.


Preparation of Plaque Forming Cholesterol, Phospholipid and Amyloid Oligomer/Aggregates:
The chemical structures of cholesterol, phospholipids, amyloid peptides and their derivatives used herein for preparation of plaque forming oligomers/aggregates has been described in previous applications (Madasamy, 2009, US Application No. 200901041211; Madasamy, 2011, PCT/US2012/066412; incorporated herein by reference). Briefly, 1 mg of lyophilized fluorescently-labeled cholesterol or cholesterol derivatives (Ex/Em=495 nm/507 nm) was solubilized in 1 mL of 100% alcohol. From this stock solution, 100 μL was taken and mixed in 900 μL of PBS. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitates, if any, and the supernatant containing mostly soluble oligomer/aggregates were used for in vitro plaque particles formation assay.
To prepare phospholipids plaque aggregates, 1 mg of fluorescently-labeled-phospholipids or its derivatives (Ex/Em=495 nm/507 nm) was solubilized in 1 mL of 100% alcohol. From this stock solution, 100 μL was taken and mixed in 900 μL of PBS. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing plaque aggregates were used for plaque array assay. The transfer of esterified cholesterol and phospholipid molecules from organic medium (alcohol) to PBS buffer lead to transformation of soluble molecules into oligomers/aggregates.
To prepare Abeta-42 oligomers/aggregates, 1 mg of fluorescently-labeled Abeta-42 peptide was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing oligomers/aggregates were used for plaque array assay. Similarly, to prepare unlabeled Abeta-42 aggregates, 1 mg of Abeta-42 peptide was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. For Abeta-28 aggregates preparation, 1 mg of Abeta-28 was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing oligomers/aggregates were used for in vitro plaque particles formation assay. For detecting unlabeled amyloid plaque particles Thioflavin S (Ex/Em=430 nm/550 nm) amyloid binding fluorescent dye (10 μg) was used.
To prepare derivatives of human Tau proteins oligomers/aggregates, 1 mg of Tau-45 Peptide (45-73 Exon 2/Insert 1 domain) was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. For Tau-74 (74-102) (Exon 3/Insert 2 domain) preparation, 1 mg of Tau-74 was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. Similarly for preparing Tau-275 (275-305) (Repeat 2 domain) 1 mg of the peptide was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. After incubation, all the samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing oligomers/aggregates were used for in vitro amyloid plaque particles formation assay. For detecting Tau plaque particles, Thioflavin S (Ex/Em=430 nm/550 nm) amyloid binding fluorescent dye (10 μg) was used in the assay.
To prepare oligomers/aggregates of human alpha-synuclein (140 amino acids polypeptide), 1 mg of the recombinant alpha-synuclein protein was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 2 hrs. After incubation, all the samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing oligomers/aggregates were used in vitro plaque particles formation assay. For detecting alpha-synuclein plaque particles Thioflavin S (Ex/Em=430 nm/550 nm) amyloid binding fluorescent dye (10 μg) was used.
Flow Cytometer Based Detection of Cholesterol, Phospholipd Amyloid Plaque Particles:
Human serum and plasma samples obtained from patients previously diagnosed for atherosclerosis related cardiovascular diseases and AD were used for examining in vitro cholesterol, phospholipid and amyloid plaque particles formation respectively. The plasma or serum samples were first centrifuged at 5,000 rpm for 5 min and the supernatants were transferred to new centrifuge tubes. Next, the supernatants were diluted in PBS to make 50% of the serum and/or plasma. The diluted samples were used for incubation with plaque forming oligomers/aggregates or self-formed plaque particles to examine in vitro plaque particle synthesis. Each assay was performed in a 200 μL reaction (100 μL of 50% plasma or serum) and 100 μL (5 μg) of the Cholesterol oligomers/aggregates and the mixtures were incubated at 37° C. for 1 hr. After the incubation, 100 μL sheath fluid was added to the mixture and the samples were used for acquisition (1-2000 events/particles for 1 min) in flow cytometer.
For detecting cholesterol plaque particles formation in the serum or plasma samples, fluorescently labeled cholesterol oligomers/aggregates prepared were used for incubation with serum samples. Each in vitro plaque particle formation assay was performed in a 200 μL reaction (100 μL of 50% plasma or serum and 100 μL (5 μg) of the fluorescently-labeled cholesterol self-formed plaque particles and the mixtures were incubated at 37° C. for 1 hr After the incubation, 100 μL sheath fluid was added to the mixture and the resulting samples were analyzed in the flow cytometer.
Next, fluorescently-labeled phospholipid plaque aggregates were prepared and used for screening the serum and plasma samples. For control experiment, fluorescently-labeled plaque aggregates were incubated in PBS and not treated with the serum. Each in vitro plaque particle formation assay was performed in a 200 μL reaction (100 μL of 50% plasma or serum and 100 μL (5 μg) of the phospholipid plaque aggregates and the mixtures were incubated at 37° C. for 1 hr. After the incubation, 100 μL sheath fluid was added to the mixture and the resulting samples were analyzed by flow cytometry.
The following assays were performed with the Abeta oligomers/aggregates. Each assay was performed in a 200 μL reaction (100 μL of diluted serum with final concentration of 25% and 100 μL (5 μg) of the unlabeled Abeta-42 or Abeta-28 aggregates and the mixtures were incubated at 37° C. for 1 hr for in vitro plaque particle formation. After incubation with diluted serum, 10 μL of Thioflavin S (Ex/Em=430 nm/550 nm) fluorescent dye (10 μg) was added and the sample was incubated for an additional 30 min. at 37° C. Following incubation, 100 μL sheath fluid was added to the mixture and the samples were used for acquisition (2000 events/particles per min) in flow cytometer.
Flow Cytometer Based Sorting and Isolation of Cholesterol and Amyloid Plaque Particles:
The goal was to isolate plaque particles synthesized in the bio-fluids in a relatively pure form to enable their subsequent use for identification of components that contribute to the plaque formation. In order to isolate cholesterol plaque particles, the plasma or serum samples obtained from atherosclerotic subjects were first centrifuged at 5,000 rpm for 5 min and the supernatants were transferred to new centrifuge tubes. Next, the supernatants were diluted in PBS to make 50% of the serum and used for incubation with cholesterol oligomers/aggregates. Each assay was performed in 1 mL reaction (800 μL of 50% plasma or serum) and 200 μL (30 μg) of the Cholesterol aggregates and the mixtures were incubated at 37° C. for 1 hr After the incubation, the mixture was directly used for sample acquisition using flow cytometer (FACS ARIA II, BD Biosciences, San Jose, Calif.). The acquisition and fluorescent dot blot analysis showed two major fractions of fluorescent cholesterol plaque particles. In order to isolate these fractions, three sets of gates were drawn in the acquisition blot to separately sort and isolate High density or high complex cholesterol particles, Low density or low complex cholesterol particles and both High and low density cholesterol particles together. The total number of cholesterol particles sorted and collected in separate tubes varied (5000 to 100,000) among serum samples of atherosclerosis subjects.
Next to isolate serum derived Abeta-42 plaque particles, the plasma or serum samples obtained from AD subjects were first centrifuged at 5,000 rpm for 5 min and the supernatants were transferred to new centrifuge tubes. Next, the supernatants were diluted in PBS to make 50% of the serum and plasma samples and used for incubation with Abeta-42 plaque oligomers/aggregates. Each assay was performed in 1 mL reaction (800 μL of 50% plasma or serum) and 200 μL (30 μg) of the Abeta-42 aggregates and the mixtures were incubated at 37° C. for 1 hr. To the mixture Thioflavin S dye (120 μg) was added and the sample was incubated for additional 30 min at 37° C. After the incubation, the mixture was directly used for sample acquisition using flow cytometer (FACS ARIA II, BD Biosciences, San Jose, Calif.). After initial detection of Abeta-42 plaque particles in the acquisition blot, gates were drawn in fluorescent dot blot to separately sort and isolate fluorescently labeled Abeta-42 particles. The total number of particles sorted and collected in separate tubes varied from 5000 to 100,000.
Similarly, to isolate serum derived Abeta-28 plaque particles, the plasma or serum samples obtained from AD subjects were first centrifuged at 5,000 rpm for 5 min and the supernatants were transferred to new centrifuge tubes. Next, the supernatants were diluted in PBS to make 50% of the serum and plasma samples and used for incubation with Abeta-28 plaque oligomers/aggregates. Each assay was performed in 1 mL reaction (800 μL of 50% plasma or serum) and 200 μL (30 μg) of the Abeta-28 aggregates and the mixtures were incubated at 37° C. for 1 hr. To the mixture Thioflavin S dye (120 μg) was added and the sample was incubated for additional 30 min at 37° C. After the incubation, the mixture was directly used for sample acquisition using flow cytometer (FACS ARIA II, BD Biosciences, San Jose, Calif.). After initial detection of Abeta-28 plaque particles, gates were drawn to separately sort and isolate fluorescently labeled Abeta-28 particles.
Next, to isolate Tau plaque particles, as performed for Abeta-42 and Abeta-28 plaque particles isolation, diluted serum samples were used for incubation separately with Tau-45, Tau-74 and Tau-275 oligomers/aggregates. Each assay was performed in 1 mL reaction (800 μL of 50% plasma or serum) and 200 μL (30 μg) of each Tau aggregates and the mixtures were incubated at 37° C. for 1 hr. To the each mixture Thioflavin S dye (120 μg) was added and the sample was incubated for additional 30 min at 37° C. After the incubation, each sample was directly used for sample acquisition using flow cytometer (FACS ARIA II, BD Biosciences, San Jose, Calif.). After initial detection of Tau plaque particles, gates were drawn in the acquisition blot to separately sort and isolate fluorescently labeled Tau plaque particles.
Similarly, to isolate alpha-synuclein plaque particles, diluted plasma or serum samples obtained from AD subjects were used for incubation with alpha-synuclein plaque oligomers/aggregates. Each assay was performed in 1 mL reaction (800 μL of 50% plasma or serum) and 200 μL (30 μg) of the alpha-synuclein aggregates and the mixtures were incubated at 37° C. for 1 hr. To the mixture Thioflavin S dye (120 μg) was added and the sample was incubated for additional 30 min at 37° C. After the incubation, the mixture was directly used for sample acquisition using flow cytometer (FACS ARIA II, BD Biosciences, San Jose, Calif.). After initial detection of alpha-synuclein plaque particles, gates were drawn in the acquisition blot to separately sort and isolate fluorescently labeled alpha-synuclein particles.
Processing of Isolated Plaque Particles for Mass Spectrescopy Based Proteomics and Glycomics Analyses:
For preparing trypsin digested peptide sequences, the isolated plaque particles were centrifuged at 10000 rpm for 5 min and the pellet containing plaque particles was resuspended in 100 μl PBS buffer. Stock solutions of digestion buffer (Ammonium Biocarbonate 50 mM), reducing buffer (DDT 100 mM) and alkylation buffer (lodoaceamide 100 mM) were prepared and stored at −20° C. for up to two months. For trypsin digestion, 100 μl of each plaque particles (2000 of Abeta-42, cholesterol, Tau and alpha-synuclein) were precipitated using cold acetone −80° C. by adding 4X's the volume. The samples were placed on dry ice for 10 min with intermittent vortex. Next, the samples were centrifuged at 4° C. at 10000 G for 10 min. Top layer of acetone was carefully removed from the tube with care not to disturb the plaque particles pellet. The samples were speed-vac for 10 min to ensure all acetone has been boiled away. The plaque particles pellet were reconstituted with 15 μl 8M Urea 100 mM Ammonium bicarbonate stock and 20 μl protease max solution. The protease max solution (Promega, Madison, Wis.) was prepared from stock by adding 60 μl 50 mM Ammonium bicarbonate and used for 3 solution digests (20 μl each). Once protease is added, the samples were vortexed on low level sonication for 10-30 minutes for ensured protein solubility followed by addition of 1.6 μl of the 500 mM DTT stock to each tube. The tubes were incubated at 55° C. for 30 minutes. The tubes were then removed and brought to the room temperature followed by addition of 3.2 μl of 1M acrylamide and incubated at room temperature for 30 minutes. To the sample, 63 μL of 50 mM Ammonium bicarbonate was added for a final volume of 98 μl and 2 μl of trypsin (10 μg, Trypsin/Lys C Mix). After mixing the solution with pipet the samples were incubated at 37° C. overnight. After the overnight digest, the tryptic digest were quenched and acidified by adding 10 μl of 50% formic acid/water stock dilution. The samples were centrifuged at 10000 G for 2 minutes and purified using stage tip and used for MS/Mass analysis.
For glycomics study, one part of the tryptic digests is used for enrichment using lectin column and the eluted glycopeptides are digested with PNGase F enzyme. at 37° C. for overnight. α-Mannosidase digestion is carried out on PNGase F-released glycans using the alpha-mannosidase enzyme, The enzyme digestion was incubated at 37° C. for 48 h with a fresh aliquot of enzyme added after 24 h and terminated by boiling for 10 min. After drying in a vacuum centrifuge the samples e re-dissolved in 10 μL 0.1% TFA and subjected Mass spec analyses using the same conditions used for the identification of plaque particles proteins
Mass Spectroscopy Analysis of Peptides for Identification of Proteins:
All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, Calif., USA; version 1.0). Sequest was set up to search the SUMS_uniprot_Human database (35847 entries) assuming the digestion enzyme strict trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 20 PPM. Propionamide of cysteine was specified in Sequest as a fixed modification. Oxidation of methionine and phospho of serine, threonine and tyrosine were specified in Sequest as variable modifications.
Scaffold (version Scaffold_4.2.1, Proteome Software Inc., Portland, Oreg.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm (Keller, A et al, 2002) with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Proteins were annotated with GO terms from NCBI (downloaded Jan. 17, 2014).
Plaque Particles Processing for Mass Spec Based Lipidomics Analysis:
Cholesterol and amyloid plaque particles sorted (5,000 particles each) were used for lipid extraction. 30 uL of each plaque particles was mixed with 200 uL MeOH to which 300 uL chloroform was added. The samples were vortexed thoroughly and incubated at room temperature for 10 minutes. Then, 120 uL H2O was added to the sample and vortexed briefly. The samples were centrifuged at 10,000 rpm for 10 min to separate phases. After centrifugation 40 uL of the lower layer was transferred to an eppendorf tube and diluted with 160 uL injection solvent (65:30:5 acetonitrile/isopropanol/water) for LC-MS analysis. The samples were used for lipidomics analysis by LC/MS on an Agilent 1260 HPLC and Bruker microTOF-Q II mass spectrometer. Full scan ms data was acquired in positive ion mode. The column was a Waters Atlantis T3 3u 2.1×100 mm with initial conditions of 60% A (60:40 acetonitrile/water with 10 mM ammonium formate)/40% B (90:10 acetonitrile/isopropanol). The flow rate was 0.4 mL/minute.
Also provided by the subject application are kits for use in practicing one or more of the above described applications embodiment. The present invention discloses a plaque array kit to aid in the diagnosis, prediction, prognosis, or detection of a plaque-associated disease such as AD and atherosclerosis. In one embodiment, the kit comprises one or more molecules for preparing plaque aggregates, or plaque oligomer as described herein along with reagents of plaque array assay and detection of plaque particles by flow cytometer or luminescence detector.
In one embodiment, the present application also discloses kits for detection of biomarkers for early diagnosis of subject sample for plaque-associate disease. The kit may include: one or more molecules for preparing plaque aggregates or plaque oligomers; materials to collect and store biological sample from a patient; reagents for incubating biological sample with the plaque aggregate or the plaque oligomer; isolating the plaque particle; detecting the presence of plaque particle; and analyzing the plaque particle through flow cytometer. Further, the kit as disclosed may also contain materials for preparing plaque particle sample for mass spectroscopy for further composition analysis.
In one embodiment instructions teaching the use of the kit according to the various methods and approaches described herein are provided. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like.
EXAMPLES
The following additional examples are offered by way of illustrations and not by way of limitation.
Example 1
Overview of the Flow Cytometer and Mass Spectroscopy Based Biomarkers Identification Method from Plaque Particles
The Example illustrated in FIG. 1 includes both a schematic diagram and steps involved in the denvelopment of plaque array method in combination with Mass spectroscopy. This method involves steps for detection, quantitation, isolation, proteomics, glycomics, lipidomics and metabolomics analysis of plaque particles.
FIG. 1 represents a schematic of the Flow cytometer and Mass spectroscopy based detection, sorting and biomarkers identification in plaque particles. Step 1, in vitro formation of insouble plaque particles in bio-fluids; step 2, Flow cytomer based identification and isolation of plaque particles; step 3A, trypsin digestion of the isolated plaque particles to prepare peptide fragments: step 3B, extraction of lipid or metabolites from plaque particles; step 4A, Mass spectroscopy (MS/MS) based proteomics and glycomics for identification of proteins/glycoproteins and step 4B, GC/MS based identification of lipids or metabolites.
Example 2
Flow Cytometer Based Detection and Quantitation of Abeta-42 Plaque Particles in AD Serum Sample
Amyloidosis is a group of more than fifteen neurodegenerative or protein aggregation diseases caused mainly by deposition of misfolded amyloid proteins or their derivatives in both intra cellular and extracellular regions of brain. The amyloid plaque related protein aggregation diseases include AD, PD, prion-mediated diseases, Tau pathies, HD, MS, type 2 diabetes and the like. Among them AD is the most common and serious neurodegenerative disease associated with progressive dementia caused mostly due to the deposition of Amyloid-beta (Abeta) peptides (Yankner 1996). Abnormal processing of the Abeta precursor protein is an early and causative event in the pathogenesis of AD (Selkoe D. J. 2003). Amyloid precursor protein (APP) undergoes a series of proteolytic cleavages culminating in production of Abeta-42 peptides and smaller peptides.
In order to examine abeta peptides based amyloid plaque particles formation in the bio-fluids, experiments were carried out using Abeta-42 oligomers/aggregates incubated in the diluted serum samples. After incubation the samples were used for analysis in Flow cytometer. The results of flow cytometry displayed herein are typically presented as one dimensional histogram on a logarithmic scale or two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range. FIG. 2A shows detection of Abeta-42 plaque particles in acquisition dot blot. FIG. 2B shows detection of fluorescence (Thioflavin S) positive Abeta-42 plaque particles and FIG. 2C shows counting of fluorescence plaque particles. In the assay, the Abeta-42 oligomers/aggregates acts as “substrate” and serum components act as “catalyzers” converting the soluble aggregates into insoluble plaque particles that are efficiently detected in the Flow cytometer. The concentrations of serum derived abeta-42 based plaque particles load in AD subjects are in the range of  ̃6000 to 64,000/mL whereas in the normal subjects the concentrations are  ̃300 to 5000/mL. The results also indicate that both specific and non-specific binding of serum ligands to the soluble plaque aggregates/oligomers lead to formation of insoluble plaque particles that are detected by flow cytometer.
FIGS. 2A, 2B and 2C shows a flow cytometer based detection of Abeta-42 plaque particles. 2A shows an acquisition dot blot analysis of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 2B shows fluorescence dot blot analysis of plaque particles (x-axis 520 nm; y-axis 560 nm). The two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range. In these plots, starting at x=0, y=0, the tick marks on the x-axes and y-axes represent fluorescent intensity of 100, 101, 102, 103 and 104 respectively and 2C shows histogram blot showing plaque particles count (Fluorescence 520 nm).
Example 3
Flow Cytometer Based Detection and Quantitation of Abeta-28 Plaque Particles in AD Serum Sample
To examine plaque particles formation in the AD patient serum sample, a different derivative of abeta peptide, Abeta-28, was used. Diluted serum sample was incubated with Abeta-28 oligomer/aggregates and after staining with Thioflavin S dye the samples were analyzed in Flow cytometer. As observed with Abeta-42 plaque particles formation described in example 2, the soluble form of aggregates are readily converted in to insoluble Abeta-28 plaque particles that are detected by the Flow cytometer. FIG. 3A shows Abeta-28 plaque particles detection in acquisition dot blot, fluorescence dot blot and histogram analysis. These results indicate that serum samples of AD patients contain molecules that can accelerate the synthesis of insoluble plaque particles from the undetectable plaque oligomers/aggregates.
FIGS. 3A, 3B and 3C shows a flow cytometer based detection of Abeta-28 plaque particles. 3A shows acquisition dot blot displays plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 3B shows fluorescence dot blot displays plaque particles (x-axis 520 nm; y-axis 560 nm). The two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range. In these plots, starting at x=0, y=0, the tick marks on the x-axes and y-axes represent fluorescent intensity of 100, 101, 102, 103 and 104 respectively and 3C shows histogram blot displaying plaque particles count (Fluorescence 520 nm).
Example 4
Flow Cytometer Based Detection of Cholesterol Particles in Atherosclerosis Patient's Serum Samples
Atherosclerosis is a chronic inflammatory cardiovascular disease caused by development of atherosclerotic plaque in the arteries. Both dyslipidemia and hypercholesterolemia are linked to initiation and progression of atherosclerosis related cardiovascular complications (Grundy S M et al, 2014; Yuasa et al, 2014). More specifically, elevated serum levels of low density cholesterol particles and reduced levels of high density cholesterol particles are known risk factors for atherosclerosis related fatal symptomatic events such as myocardial infarction and stroke (Kones R, 2011).
The following experiments were carried out to probe cholesterol particles formation in the serum samples of patients previously diagnosed with atherosclerosis. Fluorescently labeled cholesterol aggregates were incubated with diluted serum samples and the resulting sample was used for Flow cytometer analysis. FIG. 4 shows detection of cholesterol plaque particles acquired using dot blot, fluorescence dot blot and histogram blots. Two major fractions of cholesterol particles were observed in all blots. The lower density cholesterol particles (LD-Chl) or low complex cholesterol particles concentration was higher compared to higher density cholesterol particles (HD-Chl) or high complex cholesterol particles. It was observed that  ̃35% of the AD serum samples showed higher number of total cholesterol particles load ( ̃10000 to 70000/mL). As observed with abeta plaque particles formation in serum samples, cholesterol oligomer/aggregates were converted into insoluble plaque particles by serum components present in atherosclerosis patients.
FIGS. 4A, 4B and 4C show a flow cytometer based detection of cholesterol plaque particles. 4A shows acquisition dot blot displays plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 4B shows fluorescence dot blot displays plaque particles (x-axis 520 nm; y-axis 560 nm). The two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range. In these plots, starting at x=0, y=0, the tick marks on the x-axes and y-axes represent fluorescent intensity of 100, 101, 102, 103 and 104 respectively and, 4C shows histogram blot (Fluorescence 520 nm). LD-Chl refers to low density or low complex cholesterol plaque particles; HD-Chl refers to high density or high complex cholesterol plaque particles.
Example 5
Flow Cytometer Based Detection of Phospholipids Particles in Atherosclerosis Patient's Serum Samples
To further understand atherosclerotic plaque particles formation in the serum samples, phospholipid aggregates were prepared and incubated in the serum samples obtained from atherosclerosis patients. Flow cytometer analysis of the resulting samples showed two major fractions of phospholipid particles, similar to the results observed for cholesterol particles (FIG. 5).
FIGS. 5A, 5B and 5C shows detection of phospholipids plaque particles using flow cytometer. 5A shows acquisition dot blot displays plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 5B shows fluorescence dot blot analysis of plaque particles (x-axis 520 nm; y-axis 560 nm). The two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range. In these plots, starting at x=0, y=0, the tick marks on the x-axes and y-axes represent fluorescent intensity of 100, 101, 102, 103 and 104 respectively and 5C shows histogram blot analysis of (Fluorescence 520 nm) LD-Lipid refers to low density or low complex lipid plaque particles; HD-Lipid refers to high density or high complex plaque particles.
Taken together, the results of the Abeta-42, Abeta-28, cholesterol and phospholipids as plaque particles formation in the serum samples strongly indicate that the molecules present in the bio-fluids act as catalyzers to accelerate in vitro plaque particles formation. The serum samples of AD and atherosclerosis subjects contain components that catalyze in vitro formation of plaque particles from plaque forming soluble oligomers/aggregates. These results confirm our previous observation of accelerated plaque particles formation in AD and atherosclerosis serum samples compared to normal subjects (Madasamy, 2011, PCT/US2012/066412). The human serum or plasma is a complex biological fluid known to contain approximately 10,500 proteins, 107 variants of antibodies and thousands of other metabolites. However, among these large pools of molecules the specific group of molecules involved in the plaque formation is not completely identified. Accordingly, it is important to identify molecules in the bio-fluids that might play either direct or indirect role in the plaque particles formation. It is possible that the plaque forming oligomers/aggregates used herein may be entangled in a specific and non specific binding with a wide range of ligands including proteins, antibodies, lipids, carbohydrates, metals and metabolites. Identification of such biomarkers could significantly help to better understand the complex mechanism of in vivo plaque development, aid in the development of biomarkers based diagnosis, patient stratification and discovery of mechanism based anti-plaque drugs.
Example 6
Flow Cytometer Based Sorting and Isolation of Serum Derived Cholesterol Plaque Particles
Development of a rapid, sensitive and effective method for isolation of plaque particles is perquisite for biomarkers identification in the plaque particles. The preceding results described in the examples 2, 3, 4 and 5 show successful use of flow cytometer for detection, quantitation and identification of sub types of the plaque particles. In the next step, effort was made to isolate different types of plaque particles using flow cytometer based sorting method. Flow cytometer is widely used instrument to characterize, sort and isolate specific cell types (Piyasena Me. et al, 2014). Herein we describe isolation of fluorescence positive plaque particles using flow cytometer based sorting approach. First, fluorescence labeled cholesterol oligomer/aggregates were incubated in AD serum samples and used for sorting. FIG. 6A displays detection of two major fractions of cholesterol particles, FIG. 6B shows gating regions selected to sort large size cholesterol particles (5 to 60 microns in sizes) and FIG. 6C show isolated cholesterol particles. The isolated cholesterol particles (100,000) were stored at 4° C. and used for Mass spec analysis.
FIGS. 6A, 6B and 6C shows a flow cytometer based sorting and isolation of Cholesterol plaque particles. 6A shows acquisition blot displays detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 6B shows gating of fluorescence positive plaque particles population for sorting (x-axis 520 nm; y-axis 560 nm) and 6C shows acquisition blot displays sorting and isolation of gated fluorescence cholesterol plaque particles population. FSC refers to Forward scattering and SSC refers to side scattering.
Example 7
Sorting and Isolation of Serum Derived High Density and Low Density Cholesterol Plaque Particles
The results observed in example 4, suggest although the cholesterol oligomer/aggregates were prepared from a homogenous form of cholesterol when the aggregates are incubated in the serum it is converted into two major fractions of cholesterol particles. It was of interest to examine the composition of both types of cholesterol particles (high density or high complex cholesterol particles and low density or low complex cholesterol particles) separately.
FIG. 7A displays two types of cholesterol particles in acquisition blot, FIG. 7B shows double gating of cholesterol particles for sorting and isolation and FIG. 7C shows high density and low density cholesterol particles isolated separately. These cholesterol plaque particles (100,000) were stored at 4° C. until use for Mass Spec analysis.
Thus, FIGS. 7A, 7B and 7C shows a flow cytometer based sorting and isolation of High density and low density cholesterol plaque particles. 7A shows acquisition dot blot showing detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 7B shows gating of fluorescence plaque particles population selected for sorting (x-axis 520 nm; y-axis 560 nm) and 7C shows sorting and isolation of gated fluorescence population of high density and low density plaque particles (x-axis 520 nm; y-axis 560 nm).
Example 8
Flow Cytometer Based Sorting and Isolation of Abeta-42 Plaque Particles
In the next step, flow sorting experiments were repeated for isolation of Abeta-42 particles. Abeta-42 aggregates incubated in the AD serum samples were stained with Thioflavin S dye and the resulting sample was used for sorting to collect 100,000 particles. FIG. 8A shows detection of Abeta-42 particles in acquisition dot blot and FIG. 8B shows gating of large Thioflavin S positive abeta particles selected for sorting. FIG. 8C show isolated Abeta-42 plaque particles.
Thus, FIG. 8 shows a flow cytometer based sorting and isolation of Abeta-42 plaque particles. 8A shows acquisition blot displays detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 8B show gating of fluorescence positive plaque particles population for sorting (x-axis 520 nm; y-axis 560 nm) and 8C shows acquisition blot displays sorting and isolation of gated fluorescence Abeta-42 plaque particles population. FSC refers to Forward scattering and SSC refers to side scattering.
Example 9
Flow Cytometer Based Sorting and Isolation of Abeta-28 Plaque Particles
As performed earlier for isolation Abeta-42 plaque particles, AD serum sample incubated with Abeta-28 oligomers/aggregates were stained with Thioflavin dye and the resulting sample was used for Flow sorting. Unlike cholesterol particles, the fluorescence dot blot (FIG. 9C) shows only a single fraction of abeta particles population that was marked for sorting and isolation. The isolated Abeta-28 plaque particles were used for Mass spectroscopy analysis.
Thus, FIG. 9 shows a flow cytometer based sorting and isolation of Abeta-28 plaque particles. 9A shows acquisition blot displays detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 9B shows gating of fluorescence positive plaque particles population for sorting (x-axis 520 nm; y-axis 560 nm) and 9C shows acquisition dot blot displays sorting and isolation of gated fluorescence Abeta-28 plaque particles population. FSC refers to Forward scattering and SSC refers to side scattering.
Example 10
Flow Cytometer Based Sorting and Isolation of Tau Plaque Particles
In addition to Abeta peptides, Tau peptides are second major amyloid peptides found to be associated with amyloid plaques and implicated in the pathogenesis of AD related dementia (Bloom G S, 2014; Mathis Calif. et al, 2012). Hence, it was of interest to examine and compare the composition of serum derived Tau plaque particles. As described in the examples 8 and 9, AD serum samples incubated with Tau oligomer/aggregates were stained with Thioflavin S dye and the resulting samples were used for Flow sorting. As shown in FIGS. 10 A and B, the fluorescence Tau particles were gated for sorting. FIG. 10C shows isolated Tau plaque particles.
FIGS. 10A, 10 B and 10 C shows a flow cytometer based sorting and isolation of Tau (275-305) plaque particles. 10A shows acquisition dot blot showing detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis); 10B shows gating of fluorescence plaque particles population selected for sorting (x-axis 520 nm; y-axis 560 nm) and 10C shows sorting and isolation of gated fluorescence population of (275-305) plaque particles. FSC refers to forward scattering and SSC refers to side scattering.
Example 11
Flow Cytometer Based Sorting and Isolation of Alpha-Synuclein Plaque Particles
Similar to Abeta and Tau peptides, alpha-synuclein is another major amyloid protein implicated in the development of Parkinson disease. These amyloid peptides and their aggregates often found co-accumulated in the cerebral regions thus contributing to the development of amyloid related neurodegenerative disorders (Hashimoto M, 1999). In the next step Flow sorting experiments were carried out to isolate alpha-synuclein plaque particles. As performed with other amyloid peptides, alpha-synuclein aggregates were incubated with AD serum samples and after staining with Thioflavin S dye the samples were used for Flow sorting. FIGS. 11A and 11B shows detection and gating of fluorescent alpha-synuclein plaque particles and FIG. 11C shows isolated plaque particles used for Mass spec analysis.
FIG. 11 shows a flow cytometer based sorting and isolation of alpha-synuclein plaque particles. 11A shows an acquisition dot blot showing detection of plaque particles in both side scattering (y-axis) and forward scattering (x-axis) acquisition blot; 11B shows a gating of fluorescence plaque particles population selected for sorting (x-axis 520 nm; y-axis 560 nm) and 11C shows sorting and isolation of gated fluorescence population of alpha-synuclein plaque particles. FSC refers to forward scattering and SSC refers to side scattering.
Example 12
Mass Spectroscopy Based Proteomics for Identification of Proteins in Plaque Particles
The plaque particles isolated were used for identifying their composition by proteomics, glycomics, lipidomics and metabolomics approaches. For proteomics analysis, approximately 2000 particles of Abeta-42, Tau, alpha-synuclein, cholesterol L (High density cholesterol particles) and cholesterol S (Low density cholesterol particles) were taken. After trypsin digestion, purified peptide fragments were used for MS/MS analysis to generate peptide spectrum. Approximately 13778 peptide mass spectrums obtained were used for database search to identify the related proteins. Database search was carried out as described in the method section 4. Similarly, mass spec based lipidomics showed a number of lipid molecules present in the plaque particles. A few examples of Mass spectroscopy generated peptide spectrum and lipid chromatogram/spectrum are shown in FIG. 12.
FIG. 12 shows examples of Mass Spectrum data generated to deduce peptide sequences and lipid molecules. 12A shows a spectrum displays mass/charge (x-axis) and relative intensity of each peak (y-axis) the amino acid sequences of the peptide (LYHSEAFTVNFGDTEEAK) is shown at the top; 12B shows a spectrum displays mass/charge (x-axis) and relative intensity of each peak (y-axis) the amino acid sequences of the peptide (Seq ID [[#]] NO: 8) (WERPFEVKDTEEEDFHVDQVTTVK) is shown at the top and 12C shows a spectrum displays mass/charge (x-axis) and relative intensity of each peak (y-axis) the amino acid sequences of the peptide (Seq ID[[#]] NO: 9) (VFSNGADLSGVTEEAPLK) is shown at the top. 12D shows base peak chromatogram indicating number of lipid molecules in the plaque particles with (y-axis) displaying relative intensity of each peak and (x-axis) different fractions eluted over the time. 12E displays full scan mass spectra for identification of different lipids with (y-axis) displaying relative intensity of each spectrum and (x-axis) displaying mass/charge.
The database search of peptide spectrum obtained for all plaque particles revealed approximately 236 serum proteins in 188 clusters. The name of the proteins and their accession numbers are listed in Table 2. Further, analysis of the semi-quantitative binding profile of identified proteins showed that Abeta-42, cholesterol, Tau and alpha-synuclein particles have different levels of affinities to the proteins. It is important to note that some of these proteins are expected to present in the serum samples of normal subjects. However, under pathological conditions expression of these proteins might vary and they undergo post-translational modifications such as aberrant glycolylation, phophorylation, oxidation etc. Together, these factros contributing to pathogenesis of plaque related diseases including atherosclerosis and amyloidosis. These results strongly suggest that a wide range of proteins interplay in the plaque formation and their qualitative and quantitative analysis will greatly aid in the diagnosis, drug discovery and drug developments. Mass spectroscopy based measuring of expression of these proteins by multiple reaction monitoring (MRM) approach will further help to identify disease specific biomarkers in plaque development.
Example 14
Database of Proteins Identified in the Plaque Particles and Semi-Quantitative Analysis of their Binding Affinity
Table 2 shows database of proteins identified for Abeta-42, High Density cholesterol (Chl Large), Low density cholesterol (Chl small), alpha-synuclein and Tau plaque particles. Higher numbers in the semi-quantitative columns refers to stronger affinity of a particular protein and lower the number refers to weaker affinity of the protein to the plaque particles.







TABLE 2







‘Plaqueproteome’ Database showing proteins associated with test plaque particles.










Semi-quantitative analysis




of proteins binding to
Pub Med


Proteins identified from
different plaque particles
Database













plaque particles

Chl
Chl


Protein


Protein name
Abeta-42
Large
small
Synuclein
Tau
Accession #

















1.
Serum albumin
199
340
320
398
281
sp|P02768


2.
Cluster of Complement
63
73
67
71
85
sp|P01024



C3


3.
Complement C3
63
73
67
71
85
sp|P01024


4.
Serotransferrin
46
66
73
78
48
sp|P02787


5.
Apolipoprotein B-100
62
47
51
42
65
sp|P04114


6.
Cluster of Ig gamma-1
23
57
63
56
37
sp|P01857



chain C region


7.
Ig gamma-1 chain C
12
41
51
44
27
sp|P01857



region


8.
Ig gamma-2 chain C
11
12
14
10
11
sp|P01859



region


9.
Ig gamma-3 chain C
11
15
11
14
13
sp|P01860



region


10.
Cluster of Alpha-2-
32
43
43
44
45
sp|P01023



macroglobulin


11.
Alpha-2-macroglobulin
32
43
43
44
45
sp|P01023


12.
Pregnancy zone protein
4
0
2
0
0
sp|P20742


13.
Cluster of Complement
32
44
36
37
52
sp|P0C0L5



C4-B


14.
Complement C4-B
32
44
35
37
52
sp|P0C0L5


15.
Complement C4-A
28
44
36
37
50
sp|P0C0L4


16.
Cluster of Keratin, type II
171
14
20
3
24
sp|P35908



cytoskeletal 2 epidermal


17.
Cluster of Alpha-1-
28
22
21
20
28
sp|P01009



antitrypsin


18.
Alpha-1-antitrypsin
28
22
21
20
28
sp|P01009


19.
Ig kappa chain C region
11
17
21
15
15
sp|P01834


20.
Apolipoprotein A-I
19
21
21
18
14
sp|P02647


21.
Cluster of Haptoglobin
25
29
31
24
26
sp|P00738


22.
Haptoglobin
23
27
29
22
24
sp|P00738


23.
Isoform 2 of Haptoglobin-
11
15
12
14
11
sp|P00739-2



related protein


24.
Ceruloplasmin
9
20
22
21
20
sp|P00450


25.
Isoform 3 of Fibronectin
9
25
19
16
23
sp|P02751-3


26.
Cluster of Complement
4
13
15
10
11
sp|P08603



factor H


27.
Complement factor H
4
13
15
10
11
sp|P08603


28.
Cluster of Ig alpha-1
9
10
9
12
12
sp|P01876



chain C region


29.
Ig alpha-1 chain C region
9
10
8
11
12
sp|P01876


30.
Ig alpha-2 chain C region
7
9
6
10
10
sp|P01877


31.
Plasminogen
7
11
9
7
13
sp|P00747


32.
Vitamin D-binding
12
21
18
17
19
sp|P02774



protein


33.
Hemopexin
11
22
16
18
16
sp|P02790


34.
Transthyretin
14
25
21
23
13
sp|P02766


35.
Cluster of Ig mu chain C
5
10
10
9
11
sp|P01871



region


36.
Ig mu chain C region
5
10
10
8
10
sp|P01871


37.
Ig mu heavy chain disease
0
8
6
6
6
sp|P04220



protein


38.
Inter-alpha-trypsin
9
10
12
13
14
sp|P19823



inhibitor heavy chain H2


39.
Cluster of Inter-alpha-
2
20
13
12
15
sp|Q14624



trypsin inhibitor heavy



chain H4


40.
Inter-alpha-trypsin
2
20
13
12
15
sp|Q14624



inhibitor heavy chain H4


41.
Isoform 2 of Inter-alpha-
2
20
13
12
15
sp|Q14624-2



trypsin inhibitor heavy



chain H4


42.
Complement factor B
12
9
14
10
13
sp|P00751


43.
Cluster of Ig lambda-2
9
5
9
8
11
sp|P0CG05



chain C regions


44.
Ig lambda-2 chain C
9
5
7
8
10
sp|P0CG05



regions


45.
Immunoglobulin lambda-
9
5
7
8
11
sp|B9A064



like polypeptide 5


46.
Ig lambda-7 chain C
2
3
2
4
3
sp|A0M8Q6



region


47.
Ig lambda-6 chain C
5
3
3
6
5
sp|P0CF74



region


48.
Prothrombin
4
16
19
11
23
sp|P00734


49.
Alpha-1-antichymotrypsin
11
14
11
20
16
sp|P01011


50.
Gelsolin
2
8
9
6
9
sp|P06396


51.
Cluster of Kininogen-1
0
4
8
6
1
sp|P01042


52.
Kininogen-1
0
3
7
4
1
sp|P01042


53.
Isoform LMW of
0
4
8
6
1
sp|P01042-2



Kininogen-1


54.
Antithrombin-III
5
8
6
9
6
sp|P01008


55.
Apolipoprotein A-IV
5
8
9
7
10
sp|P06727


56.
Alpha-2-HS-glycoprotein
2
9
11
10
10
sp|P02765


57.
Alpha-1B-glycoprotein
4
7
7
11
6
sp|P04217


58.
Afamin
2
6
6
6
3
sp|P43652


59.
Beta-2-glycoprotein 1
4
6
6
7
5
sp|P02749


60.
Histidine-rich
7
10
9
9
12
sp|P04196



glycoprotein


61.
Inter-alpha-trypsin
7
11
7
10
18
sp|P19827



inhibitor heavy chain H1


62.
Plasma protease C1
4
7
7
9
3
sp|P05155



inhibitor


63.
Apolipoprotein E
11
7
9
8
13
sp|P02649


64.
Apolipoprotein A-II
5
8
6
7
10
sp|P02652


65.
Complement C5
0
5
3
4
1
sp|P01031


66.
Serum
2
5
6
7
3
sp|P27169



paraoxonase/arylesterase


67.
C4b-binding protein alpha
0
8
8
7
1
sp|P04003



chain


68.
Angiotensinogen
14
10
9
7
15
sp|P01019


69.
CD5 antigen-like
4
6
7
6
3
sp|O43866


70.
Zinc-alpha-2-glycoprotein
5
4
5
7
4
sp|P25311


71.
Cluster of Desmoplakin
12
0
0
0
0
sp|P15924


72.
Alpha-1-acid glycoprotein 2
9
5
7
10
9
sp|P19652


73.
Alpha-1-acid glycoprotein 1
12
9
8
7
11
sp|P02763


74.
Cluster of Hemoglobin
4
3
2
5
3
sp|P68871



subunit beta


75.
Hemoglobin subunit delta
0
0
0
2
0
sp|P02042


76.
Protein AMBP
5
5
5
5
10
sp|P02760


77.
Vitronectin
4
4
2
4
4
sp|P04004


78.
Alpha-2-antiplasmin
0
6
3
5
5
sp|P08697


79.
Hemoglobin subunit alpha
2
4
5
5
5
sp|P69905


80.
Complement C1r
0
2
4
3
4
sp|P00736



subcomponent


81.
Isoform 2 of Clusterin
4
6
4
7
5
sp|P10909-2


82.
Cluster of Ig kappa chain
2
4
4
7
4
sp|P01620



V-III region


83.
Plasma kallikrein
0
1
3
0
3
sp|P03952


84.
Complement component
0
4
6
1
4
sp|P10643



C7


85.
Cluster of Ig kappa chain
4
5
4
6
3
sp|P01625



V-IV region Len


86.
Heparin cofactor 2
0
3
1
1
6
sp|P05546


87.
Apolipoprotein D
0
2
2
1
3
sp|P05090


88.
Complement factor I
0
4
3
4
3
sp|P05156


89.
Corticosteroid-binding
0
2
3
1
1
sp|P08185



globulin


90.
Pigment epithelium-
5
4
3
4
4
sp|P36955



derived factor


91.
Ig gamma-4 chain C
7
7
11
8
11
sp|P01861



region


92.
Complement component
0
1
1
3
1
sp|P13671



C6


93.
Cluster of Ig kappa chain
5
5
1
3
2
sp|P01597



V-I region DEE


94.
Ig kappa chain V-I region
5
5
1
3
2
sp|P01597



DE


95.
Ig kappa chain V-I region
0
1
1
0
0
sp|P01612



Mev


96.
Ig kappa chain V-I region
2
1
1
0
0
sp|P01594



AU


97.
Isoform 2 of Fibrinogen
0
0
1
0
1
sp|P02671-2



alpha chain


98.
Cluster of Ig kappa chain
2
1
4
2
1
sp|P01617



V-II region TEW


99.
Complement C1s
0
3
1
1
2
sp|P09871



subcomponent


100.
Cluster of Ig heavy chain
7
5
6
4
5
sp|P01777



V-III region TEI


101.
Ig heavy chain V-III
5
4
5
3
4
sp|P01777



region TEI


102.
Ig heavy chain V-III
7
5
6
4
5
sp|P01766



region BRO


103.
Kallistatin
0
1
0
0
1
sp|P29622


104.
Retinol-binding protein 4
0
2
2
2
2
sp|P02753


105.
Isoform 2 of
0
1
1
0
2
sp|O14791-2



Apolipoprotein L1


106.
Insulin-like growth factor-
0
1
1
0
1
sp|P35858



binding protein complex


107.
Ig heavy chain V-III
2
0
1
2
4
sp|P01781



region Gal


108.
Carboxypeptidase N
0
0
1
2
1
sp|P22792



subunit 2


109.
Lumican
0
4
3
0
0
sp|P51884


110.
Immunoglobulin J chain
0
1
1
2
1
sp|P01591


111.
Apolipoprotein C-III
4
4
4
1
5
sp|P02656


112.
Leucine-rich alpha-2-
2
2
3
2
1
sp|P02750



glycoprotein


113.
Complement component
0
1
2
0
2
sp|P07358



C8 beta chain


114.
Coagulation factor XII
0
0
0
1
1
sp|P00748


115.
Complement component
0
0
1
2
2
sp|P07360



C8 gamma chain


116.
Serum amyloid A-4
2
1
1
1
4
sp|P35542



protein


117.
Complement C2
0
0
1
0
0
sp|P06681


118.
Complement component
0
3
2
0
0
sp|P07357



C8 alpha chain


119.
Isoform 2 of N-
0
4
1
2
5
sp|Q96PD5-2



acetylmuramoyl-L-alanine



amidase


120.
Serum amyloid P-
5
1
1
0
2
sp|P02743



component


121.
Ig lambda chain V-III
2
1
1
2
1
sp|P80748



region LOI


122.
Ig kappa chain V-III
0
1
0
1
0
sp|P04433



region VG


123.
Complement C1q
0
1
0
2
2
sp|P02746



subcomponent subunit B


124.
Desmoglein-1
5
0
0
0
0
sp|Q02413


125.
Filaggrin
2
0
0
0
0
sp|P20930


126.
Vitamin K-dependent
0
0
1
0
0
sp|P07225



protein S


127.
Ig heavy chain V-III
2
1
1
1
1
sp|P01767



region BUT


128.
Apolipoprotein C-II
0
0
1
1
0
sp|P02655


129.
Platelet basic protein
0
0
0
1
2
sp|P02775


130.
Ig lambda chain V-III
0
1
1
2
2
sp|P01714



region


131.
Thyroxine-binding
2
0
1
0
1
sp|P05543



globulin


132.
Ig kappa chain V-III
0
2
1
2
3
sp|P01621



region NG9


133.
Ig kappa chain V-I region
5
2
2
2
3
sp|P01613



Ni


134.
Cluster of Ig kappa chain
5
1
1
1
1
sp|P01602



V-I region HK102


135.
Ig kappa chain V-I region
5
1
1
1
1
sp|P01602



HK102


136.
Ig kappa chain V-I region
5
1
1
1
1
sp|P01596



CAR


137.
Lactotransferrin
5
0
0
0
0
sp|P02788


138.
Isoform Short of
4
0
0
0
0
sp|Q00839-2



Heterogeneous nuclear



ribonucleoprotein


139.
Apolipoprotein C-I
2
1
1
0
1
sp|P02654


140.
Hornerin
2
0
0
0
1
sp|Q86YZ3


141.
Phosphatidylinositol-
0
1
1
0
2
sp|P80108



glycan-specific



phospholipase D


142.
Tetranectin
0
0
1
0
0
sp|P05452


143.
Filaggrin-2
4
0
0
0
0
sp|Q5D862


144.
Isoform 2 of Ficolin-3
0
0
0
1
0
sp|O75636-2


145.
Cluster of Ig heavy chain
7
2
3
1
3
sp|P01764



V-III region VH26


146.
Ig heavy chain V-III
7
2
3
1
3
sp|P01764



region VH26


147.
Ig heavy chain V-III
5
0
2
0
2
sp|P01765



region TIL


148.
Complement C1q
0
1
1
1
2
sp|P02747



subcomponent subunit C


149.
Cluster of Platelet factor 4
2
1
0
1
1
sp|P02776


150.
Platelet factor 4
2
1
0
1
1
sp|P02776


151.
Platelet factor 4 variant
0
1
0
1
0
sp|P10720


152.
Cluster of Actin,
0
3
0
0
0
sp|P60709



cytoplasmic 1


153.
Actin, cytoplasmic 1
0
3
0
0
0
sp|P60709


154.
Complement component
0
1
1
1
0
sp|P02748



C9


155.
Junction plakoglobin
2
0
0
0
0
sp|P14923


156.
Ig heavy chain V-III
2
1
1
1
1
sp|P01763



region WEA


157.
Cluster of Isoform B of
0
3
3
1
0
sp|P23142-3



Fibulin-1


158.
Isoform B of Fibulin-1
0
3
3
1
0
sp|P23142-3


159.
Isoform C of Fibulin-1
0
1
2
1
0
sp|P23142-4


160.
Protein S100-A7
2
0
0
0
0
sp|P31151


161.
Isoform 2 of Glycogen
4
0
0
0
0
sp|P49841-2



synthase kinase-3


162.
Cluster of Nucleolar RNA
2
0
0
0
0
sp|Q9NR30



helicase 2


163.
Nucleolin OS = Homo
4
0
0
0
0
sp|P19338



sapiens GN = NCL PE = 1



SV = 3


164.
Protein-glutamine
2
0
0
0
0
sp|Q08188



gamma-



glutamyltransferase E


165.
Ig heavy chain V-III
0
0
1
1
1
sp|P01780



region JON


166.
Ig kappa chain V-I region
2
2
1
2
2
sp|P01611



Wes


167.
Cluster of Interleukin
4
0
0
0
0
sp|Q12906



enhancer-binding factor 3


168.
Interleukin enhancer-
4
0
0
0
0
sp|Q12906



binding factor 3


169.
Isoform 2 of Spermatid
2
0
0
0
0
sp|Q96SI9-2



perinuclear RNA-binding



protein


170.
Isoform 2 of Annexin A2
2
0
0
0
0
sp|P07355-2


171.
Isoform 1B of
4
0
0
0
0
sp|Q08554-2



Desmocollin-1


172.
Coagulation factor XIII B
0
0
1
0
0
sp|P05160



chain


173.
Cluster of Ig heavy chain
4
2
3
1
0
sp|P01770



V-III region NIE


174.
Ig heavy chain V-III
0
1
1
1
0
sp|P01770



region NIE


175.
Ig heavy chain V-III
4
2
3
1
0
sp|P01779



region TUR


176.
Cluster of Histone H1.2
2
0
0
0
0
sp|P16403


177.
Histone H1.2
2
0
0
0
0
sp|P16403


178.
Histone H1.3
2
0
0
0
0
sp|P16402


179.
Isoform 2 of Arginase-1
2
0
0
0
0
sp|P05089-2


180.
Caspase-14
5
0
0
0
0
sp|P31944


181.
Ig lambda chain V-IV
0
0
1
0
1
sp|P01717



region Hil


182.
Ig kappa chain V-I region
2
2
1
1
2
sp|P04430



BAN


183.
Cathepsin D
5
0
0
0
0
sp|P07339


184.
Complement factor H-
0
2
1
0
1
sp|Q03591



related protein 1


185.
Galectin-3-binding protein
0
1
0
0
0
sp|Q08380


186.
Ig heavy chain V-I region
2
1
1
2
1
sp|P01743



HG3


187.
Heterogeneous nuclear
2
0
0
0
0
sp|O43390



ribonucleoprotein R


188.
Lysozyme C
4
0
0
0
0
sp|P61626


189.
RNA polymerase-
2
0
0
0
0
sp|Q6PD62



associated protein CTR9


190.
40S ribosomal protein S8
4
0
0
0
0
sp|P62241


191.
Isoform APP639 of
4
0
0
0
0
sp|P05067-



Amyloid beta A4 protein





10


192.
Phosphatidylcholine-
0
1
0
1
0
sp|P04180



sterol acyltransferase


193.
Apolipoprotein F
0
0
1
2
1
sp|Q13790


194.
Coagulation factor X
0
0
1
0
0
sp|P00742


195.
Glyceraldehyde-3-
5
0
0
0
0
sp|P04406



phosphate dehydrogenase


196.
Parafibromin
2
0
0
0
0
sp|Q6P1J9


197.
Isoform 2 of C4b-binding
0
0
1
0
0
sp|P20851-2



protein beta chain


198.
Cluster of Nuclease-
4
0
0
0
0
sp|P67809



sensitive element-binding



protein 1


199.
Nuclease-sensitive
4
0
0
0
0
sp|P67809



element-binding protein 1


200.
Isoform Tau-G of
0
0
0
0
2
sp|P10636-9



Microtubule-associated



protein tau


201.
Tubulin alpha-1A chain
0
3
0
0
0
sp|Q71U36


202.
Serpin B3
2
0
0
0
0
sp|P29508


203.
Immunoglobulin lambda-
0
0
1
0
0
sp|P15814



like polypeptide 1


204.
Ig kappa chain V-III
0
0
1
0
2
sp|P06311



region IARC/BL41


205.
Thioredoxin
2
0
0
0
0
sp|P10599


206.
Ig kappa chain V-III
0
1
1
0
2
sp|P01624



region POM


207.
Cluster of Histone H2A
0
0
1
0
0
sp|P04908



type 1-B/E


208.
Isoform 2 of Histone
0
0
1
0
0
sp|Q9BTM1-2



H2A.J


209.
Cluster of RNA
2
0
0
0
0
sp|Q8N7H5



polymerase II-associated



factor 1 homolog


210.
40S ribosomal protein S4, X
4
0
0
0
0
sp|P62701


211.
Isoform 2 of Protein SET
4
0
0
0
0
sp|Q01105-2


212.
Isoform 2 of RNA-
2
0
0
0
0
sp|Q14498-2



binding protein 39


213.
Selenoprotein P
0
0
0
1
0
sp|P49908


214.
Isoform 2 of
4
0
0
0
0
sp|P06748-2



Nucleophosmin


215.
60S ribosomal protein
2
0
0
0
0
sp|P27635



L10


216.
Ig heavy chain V-III
4
1
2
0
1
sp|P01776



region WA


217.
Dermcidin
2
0
0
0
0
sp|P81605


218.
60S ribosomal protein
2
0
0
0
0
sp|Q07020



L18


219.
Serpin B12
2
0
0
0
0
sp|Q96P63


220.
Carboxypeptidase N
0
0
0
0
1
sp|P15169



catalytic chain


221.
Tubulin beta-4B chain
0
0
0
0
5
sp|P68371


222.
Putative tubulin beta
0
0
0
0
6
sp|A6NKZ8



chain-like protein


223.
Peroxiredoxin-1
5
0
0
0
0
sp|Q06830


224.
Myosin-6
17
0
0
0
0
sp|P13533


225.
Myosin-13
17
0
0
0
0
sp|Q9UKX3


226.
Myosin-1
22
0
0
0
0
sp|P12882


227.
Isoform MBP-1 of Alpha-
7
0
0
0
0
sp|P06733-2



enolase


228.
Tropomyosin alpha-3
5
0
0
0
0
sp|P06753-3



chain


229.
Guanine deaminase
5
0
0
0
0
sp|Q9Y2T3-3


230.
Nucleoside diphosphate
5
0
0
4
0
sp|P15531-2



kinase A


231.
GTP-binding nuclear
5
0
0
0
0
sp|P62826



protein


232.
Catalase
12
0
0
0
0
sp|P04040


233.
Apolipoprotein M
18
0
0
0
0
sp|O95445


234.
Actin, cytoplasmic 1
49
0
0
27
0
sp|P60709


235.
POTE ankyrin domain
18
0
0
18
0
sp|Q6S8J3



family member E


236.
Spectrin
12
5
5
24
6









Table 2 shows database of “plaqueproteome” identified for Abeta-42, High Density cholesterol (Chl Large), Low density cholesterol (Chl small), alpha-synuclein and Tau plaque particles. The database of biomarkers described herein will be used to select one or more biomarkers for developing assays for selective identification of each plaque particles. Such selective plaque test assays will be used for diagnosis, drug screening and drug development purposes.
Example 13
Comparative Analysis of Identified Peptides and Proteins in Different Plaque Particles
Interestingly, comparative analysis of proteins identified in all plaque particles reveals that a large number of proteins, shown in the intersection of Venn diagram, are overlapped in all plaque particles (FIG. 13A, 13B, 13C, 13D). In addition, number of specific proteins identified for all plaque particles are shown in the left, right and bottom of Venn diagram. Significantly, it was observed that abeta-42 plaque particles generated in the AD serum samples contain more specific proteins compared to age-matched control together, these results suggest that proteins identified in this study might play key role in modulating “plaque formation system”. Previously, multiple clinical and proteomics studies been carried out identify biomarkers from whole serum or plasma samples of atherosclerosis and AD (Yuasa Y et al, 2014; Wang Q et al, 2005; Sui X et al, 2014; Minjarez B et al, 2014). Conversely, the method described herein, instead of analyzing total proteins of bio-fluids, is more specific in identifying molecules or biomarkers that interplay in the plaque formation system.
FIG. 13 shows a comaparative analysis of common and specific proteins identified in plaque particles. 13A shows a Venn diagram showing number of proteins identified in all three amyloid plaque particles (intersection of three sets) and specific proteins identified (shown in left, right and bottom of three sets) in a particular type of plaque particle. 13B shows a Venn diagram showing number of proteins shared (intersection of three sets) among cholesterol and a beta 42plaque particles and specific proteins identified (shown in left, right and bottom of three sets) in a particular type of plaque particles. Cholesterol L refers to High density cholesterol paticles and Cholesterol S refers to Low density cholesterol particles. C, diagram showing number of proteins identified among choelerol, and Tau plaque particles (intersection of three sets) and specific proteins identified (shown in left, right and bottom of three sets) in a particular type of plaque particle. D, Venn diagram showing number of proteins shared (intersection of three sets) among cholesterol and alpha-synuclein plaque particles and specific proteins identified (shown in left, right and bottom of three sets) in a particular type of plaque particles.
Example 14
Analysis of Protein Clusters and their Role in Multiple Biochemical and Cellular Pathways
The proteins identified in various plaque particles are known to be involved in the function of multiple biochemical pathways. The database analysis of these proteins showed that they are associated with 188 clusters (FIG. 14) with five major clusters among them are metabolic, immune, complement, proteases and apolipoprotein pathways. Under normal conditions these proteins play key roles in the functions of these biochemical pathways. However, under pathological conditions expression of these proteins may be up or down regulated leading to biochemical and cellular dysfunctions causing serious plaque related medical conditions such as myocardial infarction and dementia. To probe the post-translational glycosylation of proteins, the search of plaqueproteome database described here revealed Nex and O=Hex NAc modification in multiple proteins.
Example 15
Identification of Plaque Specific Antibodies for Therapeutic and Diagnosis Applications
Human immune system plays key role in modulating origin and progression of atherosclerotic and amyloid plaques. Antibodies are involved in the endogenous clearance of pathogenic atherosclerotic or amyloid aggregates/oligomers and defective clearance of plaque aggregates leads to accelerated accumulation of plaques in the arteries. Accordingly, the search of our ‘plaqueproteomics” database helped us to identify the following plaque specific antibodies and their fragments.







TABLE 3







Abeta-42 specific antibody sequences involved in the


Abeta plaque particles formation:









Sequence




Number
Name
Sequence





SEQ ID NO: 10
Lambda chain-7 C
GQPKAAPSVTLFPPSSEELQANKATLVCLVSDF



region
YPGAVTVAWKADGSPVKVGVETTKPSKQSNN




KYAASSYLSLTPEQWKSHRSYSCRVTHEGSVEK




TVAPAECS


 









SEQ ID NO: 11
Kappa chain V-III
MEAPAQLLFLLLWLPDTTREIVMTQSPPTLSLSP



region VH
GERVTLSCRASQSVSSSYLTWYQQKPGQAPRLL




IYGASTRATSIPARFSGSGSGTDFTLTISSLQPEDF




AVYYCQQDHNLP


 


SEQ ID NO: 12
Heavy chain V-III
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRVLSS



region
WVRQAPGKGLEWVSGRLNASSNLHFAVSAQGRF




TISRNDSKNTLYLQMLSLQAZBTALYYCARLLSVY




AVAFDVWGQGTKVS


 


SEQ ID NO: 13
Ig gamma-2 chain C
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP



region
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV




PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE




CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCV




VVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ




FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP




APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL




TCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDS




DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN




HYTQKSLSLSPGK


 


SEQ ID NO: 14
Ig gamma-1 chain C
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP



region
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV




PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT




HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT




CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE




EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK




ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ




VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV




LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA




LHNHYTQKSLSLSPGK
















TABLE 4







Tau specific antibody sequences involved in the Tau plaque


particles formation:









Sequence




Number
Name
Sequence





SEQ ID NO: 15
Ig kappa chain
EIVLTQSPGTLSLSPGERATLSCRAALLSSRGYLAWY



region V-III*
QQKPGQAPRLLMYGASSRATGIPDRFSGSGSGTDFTL




TISRLEPEDFAVYYCQQYGSSPRSFGQGTKVEIKR


 









SEQ ID NO: 16
Ig kappa chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSNSFLAWYQ



region V-III (2)
QKPGQAPRLLIYVASSRATGIPDRFSGSGSGTDFTLTIS




RLEPEDFAVYYCQQYGSSPSTFGQGTVELKR





*Ig kappa chain region V-III with unique peptide sequences determining Tau specific binding.













TABLE 5







Alpha-synuclein specific antibody sequences 


involved in the plaque particles formation:









Sequence




Number
Name
Sequence





SEQ ID NO: 17
Ig kappa chain
DVQMTQSPSSLSAVGDRVIITCRASQSSV



region V-I
DYLNWYQQKPGKAPKLLIFDTSNLQSGV




PSRFSGGRSGTDFTLTISSLQPDDFATYYC




QQSYTNPEVTFGGGTTVDIKR


 


SEQ ID NO: 18
Ig kappa chain
DVVMTQSPLFPVTLGEPASIQCRSSQSLVYB



V-II region
GBTYLBWYLQKPGSPELLIYLSSYRDSGVPD




RLSDSGSGTDFTLKITRVQAEDVGVYYCMQ




ATZSPYTFGQGTKLZIKR
















TABLE 6







Cholesterol (High density and low density) specific antibody


sequences involved in the cholesterol plaque particles


formation:









Sequence




Number
Name
Sequence





SEQ ID NO: 19
Ig kappa V-I region
DIQMTQSPSTLSASVGDRVAIRTCRASQNI




SSWLAWYQQKPGKAPKVLIYKSSSLESGV




PSRFSGSGSGTDTDFTLTISSLZPBBFATYYC




QQYNTFFTGPGTVDIKR


 









SEQ ID NO: 20
Heavy chain V-III
EVQLLESGGGLVQPGGSLRLSCAASGFSFS



region
TDAMYWVRQAPGKGLEWVAWKYQEASN




SHFADTVNRGFTISRNDSKNTLYLQMNRLE




AZBTAVYYCARFRQPFVQFFDVFGQGTLVT


 


SEQ ID NO: 21
Ig heavy chain V-III
QVKLVQAGGGVVQPGRSLRLSCIASGFTF



region
SNHGMHWVRQAPGKGLEWVAVIWYNGS




RTYYGDSVKGRFTISRNDNSKRTLYMZMN




SLRTEDTAVYYCARDPDILTAFSFDYWGQG




VLVTVSS









The human antibody fragments identified here are suitable for insertion/modification into the gene encoding full length human antibody and the resulting constructs can be used to express respective functional antibodies. These novel antibodies can be successfully used as therapeutics to treat plaque related diseases in affected patients of atherosclerosis and AD. In addition, they can be used as lead molecules for further optimization to improve their specificity and efficacy. The resulting optimized antibodies have multiple applications including use as therapeutics and for diagnosis of plaque related diseases.
In conclusion, the flow cytometer based plaque array method in combination with Mass spectroscopy has been successfully used to identify compositions of various plaque particles that are related cardiovascular and neurodegenerative diseases. The ex vivo plaque formation model system described herein could be used to discover novel therapeutic molecules that modulate the process of atherosclerotic and amyloid plaque particles formation. In addition, these biomarkers identified have multiple applications including understanding mechanism of plaque development, clinical diagnosis of plaque related diseases, patient profiling, personalized medicine, companion diagnosis, pre-clinical studies in animals, drug discovery and drug developments.
The invention has been described using exemplary preferred embodiment. However, for those skilled in this field, the preferred embodiment can be easily adapted and modified to suit additional applications without departing from the spirit and scope of this invention. Thus, it is to be understood that the scope of the invention is not limited to the disclosed embodiment. On the contrary, it is intended to cover various modifications and similar arrangements based upon the same operating principle. The scope of the claims, therefore, should be accorded the broadest interpretations so as to encompass all such modifications and similar arrangements. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

## Claims

1. An in vitro method of synthesizing an insoluble plaque particle, comprising:
a) preparing a soluble amyloid plaque aggregate;
b) contacting a serum sample from a subject with the soluble amyloid plaque aggregate to form the insoluble plaque particle that is detectable in the serum sample of a patient;
c) detecting and quantifying the insoluble plaque particle using flow cytometer that do not detect the soluble amyloid plaque aggregate in the serum sample of a patient;
d) isolating the insoluble plaque particle; and
e) analyzing the insoluble plaque particle to identify composition using mass spectrometry for stratifying the specific insoluble plaque particle.

2. An in vitro method of screening for a candidate agent to treat a plaque associated disease, comprising:
a) preparing a soluble amyloid plaque aggregate in vitro wherein the plaque aggregate is linked to a detectable signal;
b) mixing a serum sample from a subject with the soluble amyloid plaque aggregate to form an insoluble plaque particle in an accelerated form outside the body;
c) adding a candidate agent to the serum sample before and after mixing with the soluble amyloid plaque aggregate to form the insoluble plaque particle;
d) isolating and detecting the insoluble plaque particle with and without the candidate agent using flow cytometer and collecting an isolated sample of the plaque particle for each serum sample;
e) analyzing and identifying a biomolecule in the isolated sample of the insoluble plaque particle using mass spectrometry; and
f) analyzing and identifying of the biomolecule identified in the plaque particle isolated from the serum sample not in contact with the candidate agent to the sample of insoluble plaque particle to screen the candidate agent as an anti-plaque agent.

3. The method of claim 2, wherein the candidate agent is an Abeta-42 antibody.