EP3464339A1

EP3464339A1 - B-type natriuretic peptide proteolytic assay for cardiovascular disease risk assessment

Info

Publication number: EP3464339A1
Application number: EP17807518.0A
Authority: EP
Inventors: Shenyan ZHANG; Koen RAEDSCHELDERS; Jennifer Van Eyk
Original assignee: Cedars Sinai Medical Center
Current assignee: Cedars Sinai Medical Center
Priority date: 2016-06-03
Filing date: 2017-06-01
Publication date: 2019-04-10
Also published as: WO2017210488A1; EP3464339A4

Abstract

Described herein are methods for assessing the risk of cardiovascular disease in a subject in need thereof by detecting the presence of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein the presence of one or more cleavage products is indicative of an increased risk of the subject developing cardiovascular disease.

Description

B-TYPE NATRIURETIC PEPTIDE PROTEOLYTIC ASSAY FOR

CARDIOVASCULAR DISEASE RISK ASSESSMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 62/345,595, filed on June 3, 2016.

FIELD OF THE INVENTION

[0002] Described herein are methods for assessing risk of cardiovascular diseases.

BACKGROUND

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] BNP is a biologically active circulating hormone whose concentration is routinely used in the diagnosis of heart failure. There are two clinical assays NT-proBNP and BNP. NT-proBNP is a large fragment generated from the pre-hormone when BNP 1-32 is cleaved. BNP 1-32 has been reported to be further cleaved in plasma (FIG. 1). However, antibody- based methods cannot distinguish those proteolytic variants.

[0005] Heart disease continues to persist as a major cause of worldwide mortality, which underscores the urgent need for improved diagnostic and risk stratification tools. B-type Natriuretic Peptide (BNP) is a key biomarker whose quantitative analysis is used to clinically assess heart failure. Physiological plasma BNP levels reflect a state of dynamic equilibrium, in which the prohormone proBNP is cleaved and secreted by cardiomyocytes, and actively processed and degraded in plasma by at least 3 known peptidases, neutral endopeptidase (NEP), dipeptidylpeptidase IV (DPPIV), and insulin degrading enzyme (IDE) potentially in conjunction with additional putative enzymes. While plasma sampling eliminates the input of newly secreted BNP, the catabolic and processing steps that occur in plasma remain intact. The length and conditions of sample storage upstream of BNP analysis is therefore an important variable. Nevertheless, the extent to which BNP processing occurs in plasma as well as its relationship with heart disease remains poorly understood. Although routine clinical analysis of BNP is performed by immunoassays, it is challenging to use such techniques to account for cleaved or alternatively modified peptidoforms whose characterization may reveal important diagnostic and prognostic insights.

[0006] The absence of quantitative methods capable of identifying cleavage products is a bottleneck that obscures clinically important functional differences. The inventors have built an assay that monitor formation of two or more fragments of BNP and also can determine the enzymatic activity which should better reflect the biological status of patients

SUMMARY OF THE INVENTION

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0008] In various embodiments, the present invention provides a method, for determining the risk of developing cardiovascular disease in a subject, comprising: obtaining a biological sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the biological sample; and detecting the presence of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein the presence of one or more cleavage products is indicative of an increased risk of the subject developing cardiovascular disease. In some embodiments, the method further comprises selecting one or more treatments for the subject if the increased risk of developing cardiovascular disease is determined. In some embodiments, the natriuretic peptides are any one or more of Brain natriuretic peptide (BNP), Atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) or combinations thereof. In some embodiments, the proteases are any one or more of neutral endopeptidase, dipeptidylpeptidase IV, insulin degrading enzyme or combination thereof. In some embodiments, the sample is plasma, blood, or serum. In some embodiments, the period of time is up to 1 hour. In some embodiments, the period of time is up to 14 hours. In some embodiments, the cleavage products are any one or more of BNP 3-30, BNP 3-29, BNP 3-32, BNP 1-30 or combinations thereof. In some embodiments, the cleavage products are any one or more of BNP 3-30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 5-29, BNP 4-29, BNP 1-28, BNP 1-29, BNP 4-31, BNP 4-32 or combinations thereof. In some embodiments, the cleavage products are any one or more of BNP 3-30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 1-29, B P 1-28, B P 2-31, B P 3-30, B P 4-30, BNP 4-29, B P 4-27, BNP 5-32, BNP 5-31, BNP 5-29, BNP 4-32, BNP 4-31, or combinations thereof. In some embodiments, the cleavage products are any one or more of 30-32, 25-30, 20-25, 25- 32, 15-20, 10-15, 5-10, 10-20 or 20-30 consecutive amino acids of the natriuretic peptides. In some embodiments, the cleavage products are not modified. In some embodiments, the cleavage products are modified. In some embodiments, the modification is oxidation at the methionine residue. In some embodiments, the cleavage products are detected using any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography mass spectrometry (LC-MS), high pressure liquid chromatography mass spectrometry (HPLC- MS), or combinations thereof. In some embodiments, the quantity of one or more natriuretic peptides added to the sample is about any one or more of lOng^L, 50ng^L, 75ng^L, lOOng^L, 125ng^L, 150ng^L, 175ng^L, 200ng^L, 225ng^L, 250ng^L, 275ng^L, 300ng^L, 350ng^L, 375ng^L, 400ng^L, 450ng^L, 475ng^L, 500ng^L or combinations thereof. In some embodiments, the cardiovascular disease is heart failure, arterial fibrillation or combination thereof. In some embodiments, the method further comprises comparing the presence of one or more cleavage products of the one or more natriuretic peptides from the subject to the presence of one or more cleavage products of the one or more natriuretic peptides from a reference sample. In some embodiments, the method further comprises making an assessment of the subject based on the comparison, wherein the assessment is a determination of the risk of developing cardiovascular disease. In some embodiments, the reference sample is obtained from a healthy subject. In some embodiments, the reference sample is obtained from a subject that has been treated for the cardiovascular disease. In some embodiments, the reference sample is obtained from the subject at an earlier point in time. In some embodiments, the reference sample is obtained from the subject before the subject is treated for the cardiovascular disease. In some embodiments, the method further comprises detecting the presence of one or more natriuretic peptides over a period of time.

[0009] As opposed to current assays that measure BNP as a marker for cardiovascular disease severity, our invention measures the activity of at least three proteases present in plasma (neutral endopeptidase, dipeptidylpeptidase IV, and insulin degrading enzyme) by measuring the extent to which they cleave BNP 1-32 into its product fragments. Plasma proteases that affect the relative amounts of BNP cleavage products can modulate the relative strength with which BNP induces signaling. As such, their collective activities represent a fundamental mechanism of deleterious signaling in cardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0011] FIG. 1 depicts in accordance with various embodiments of the invention, a schematic representation of B-type natriuretic peptide structure, cleavage processing, and degradation. ProBNPl-108 and signal peptide, cleaved to NT-proBNPl-76 and the active hormone BNP1-32. The solid black line with the scissors pictogram indicates the processing site of the enzymes Furin and Corin. Blue and green lines with scissor pictograms show the processing site on BNP that occur in the peripheral circulation as a result of three known circulating peptidases: neutral endopeptidase (NEP), dipeptidylpeptidase IV (DPPIV), and insulin degrading enzyme (IDE).

[0012] FIG. 2 depicts in accordance with various embodiments of the invention, the sequences of pre-proBNP, proBNP, NT-proBNP, and BNP itself, along with residues putatively modified by O-glycosylation, and known epitope regions targeted by detection antibodies.

[0013] FIG. 3 depicts in accordance with various embodiments of the invention, an overview of the materials and methods, as well as a representative time program of the CE separation with 3 sample segments injected by multi-segment injection.

[0014] FIG. 4 depicts in accordance with various embodiments of the invention, a schematic diagram of the CESI-MS interface and multi-segment injection (MSI), alongside the mathematic separation principle that underscores CE-based separations.

[0015] FIG. 5 depicts in accordance with various embodiments of the invention, the Total Ion Chromatogram of the effect of three spacer conditions to facilitate multi-segment injection with CESI-MS using standard BNPi-32 dissolved in water. 10%HAc was used as spacer to separate samples injected in one run. Spacer was adjusted by injection time and pressure.

[0016] FIG. 6 depicts in accordance with various embodiments of the invention, the Total Ion Chromatogram upon injecting 2, 3, 4, or 5 segments on the within one CESI-MS run. [0017] FIG. 7 depicts in accordance with various embodiments of the invention, the reproducibility of three sequentially injected standard samples of recombinant B Pi_-32, as depicted by the overlaid electropherograms of five separate experiments conducted by Capillary Electrophoresis.

[0018] FIG. 8A - FIG. 8B depicts in accordance with various embodiments of the invention, the reproducibility of the CESI-MS with multi-segment injection using (FIG. 8A) the intra-run and inter-run peak area CV of B Pi_-32 of successive CE-MS experiments with three-segment multi-segment injection using standard B Pi_-32 dissolved in water, and (FIG. 8B). The inter-run migration time for each of the three segments within a run of standard B Pi_-32 dissolved in water.

[0019] FIG. 9 depicts in accordance with various embodiments of the invention, the extracted ion chromatogram of B Pi_-32 (+5 charge state) by injecting 5 different concentrations of recombinant B Pi_-32 standard solution within one CESI-MS experiment using 5-segment multi-segment injection.

[0020] FIG. 10A - FIG. 10B depicts in accordance with various embodiments of the invention, (FIG. 10A) an initial calibration curve of B Pi_-32 by hydrodynamically injecting 5 segments consisting of different recombinant B Pi_-32 standard solution concentrations for CESI-MS analysis by 5-segment multi-segment injection. Each CESI-MS run produced a complete set of peaks for the five concentrations, and the experiment was performed in triplicate (n=3 runs) (FIG. 10B). The reproducibility of the 5-point B Pi_-32 calibration curves as measured by CESI-MS with five segment multi-segment injection (N=3).

[0021] FIG. 11 depicts in accordance with various embodiments of the invention, the total ion chromatogram, the extracted ion chromatograms of B Pi_-32 B P_3-32, and B P_{3 -2}9, and the MS spectrum of B Pi_-32 spiked into patient serum (patient age>40). Unlike control serum, an unknown peak of m/z 708 was significantly increased in patient serum.

[0022] FIG. 12A - FIG. 12B depicts in accordance with various embodiments of the invention, (FIG. 12A) the comparison of the MS² spectrum of the unknown peak 708 and the in silica predicted MS² spectrum of B P_{3 -2}9; and (FIG. 12B) daughter ion mapping of B P_3- ₃₂ fragments.

[0023] FIG. 13A - FIG. 13B depicts in accordance with various embodiments of the invention, the time-dependent profile curves of B Pi_-32, B P_3-32 and BNP_{3 -2}9 in two different human samples. (FIG. 13A) Control human serum. (FIG. 13B) Patient (>40 years old) after spiked (adding) in serum sample, BNPi_-32 was rapidly cleaved in both group, however the B P_{3 -}29 profile showed a significant increase beyond the control.

[0024] FIG. 14 depicts in accordance with various embodiments of the invention, the reproducibility of BNPi_-32 electropherograms of 5-segment electrokinetically injected multi- segment injection runs. The overlaid total ion chromatogram of four experiments, each consisting of 5 segments from recombinant B Pi_-32 protein in water (250ng/mL). Intra-run reproducibility was assessed by comparing individual MSI segments within a run, while Inter-run reproducibility was assessed by comparing the same peak across four successive runs, both with respect to migration times (table, top left table) and peak area (table, bottom left). Segments were injected in 3 minute intervals within each run, and successive runs were performed at lhr intervals.

[0025] FIG. 15 depicts in accordance with various embodiments of the invention, the BNP dilution curve. The calibration curve is produced from three separate CESI-MS runs with electrokinetically injected multi-segment injection, each consisting of five segments of increasing recombinant BNPi_-32 concentrations separated by a background electrolyte spacer. Curve is based on all data points (mean +/-Std Dev).

[0026] FIG. 16 depicts in accordance with various embodiments of the invention, Enzymatic profile of BNP. Profile of BNPi_-32 (250ng^L) pulsed into 5 different plasma dilutions (plasma:total volume). Samples were incubated inside the CE instrument at 25°C and analyzed every hour for a sequence of 12 successive runs, each consisting of 5 simultaneously analyzed MSI segments representing BNPi_-32 from every plasma dilution. No quantifiable BNPi_-32 peak was detected beyond 4hrs in the 1 :5 plasma dilution.

[0027] FIG. 17A - FIG. 17B depicts in accordance with various embodiments of the invention, Enzymatic proteolysis curves of BNP peptidoforms. Simultaneous profiling of five peptidoforms from 20 consecutive CESI-MS runs sampled after an initial 250ng^L pulse of BNPi_-32 into a plasma. (FIG. 17A) depicts representative electropherograms of the total ion chromatogram and extracted ion chromatogram for BNPi_-32, BNP_3-32, BNP_{3 -2}9, BNPi_-3o and BNP_{3 -3}o. We extracted the accurate MS¹ spectra of the most abundant charge state for each profiled BNP peptidoform: BNPi_-32 with 5+ charge, BNP_3-32 with 5+, BNP_{3 -2}9 with 4+ charge, BNPi_-3o with 5+ charge, and BNP_{3 -3}o with 5+ charge. (FIG. 17B) depicts the time course profile of BNPi_-32 and each peptidoform as individual peak areas over a total of 14hrs post- pulse (n=3). Note: Peaks below the quantitative threshold of 5e5 could not be accurately quantified and were excluded.

[0028] FIG. 18 depicts in accordance with various embodiments of the invention, the effect of Heparin versus EDTA plasma. Proteolytic profiles of exogenous BNPi_-32 (250 ng^L) pulsed into lOx diluted EDTA, Heparin, and artificial plasma using neutral-coated CESI-MS with MSI (n=5, each). Samples were incubated on the CE autosampler at 25°C for 18 min prior to selective electrokinetic injection of BNP peptidoforms using MSI with 3 min intervals (5 segments per run). Datapoints depict mean +/- Std Dev lines of best fit reflect quadratic nonlinear regression with 95 % CI.

[0029] FIG. 19 depicts in accordance with various embodiment of the invention, the concept of our invention. Namely, we simultaneously analyze BNPi_-32, BNPi_-30, BNP_3-32, BNP_3-3o, and BNP_{3 -2}9 from the sequential analyses of a sample into which BNPi_-32 is pulsed.

[0030] FIG. 20 depicts in accordance with various embodiments of the invention, the known physiological steps involved in BNPi_-32 secretion, as well as the known circulating enzymes involved in its further cleavage to ten putative cleavage peptidoforms, namely Neutral Endopeptidase (NEP), Dipeptidyl peptidase IV (DPPIV), and Insulin Degrading Enzyme (IDE).

[0031] FIG. 21 depicts in accordance with various embodiments of the invention, the analytical requirements identified at the outset of the invention process (left) and the analytical setup used to address these (Right). Conclusion: linking Capillary Electrophoresis with Mass Spectrometry addresses the simplified sample preparation and intact proteins inherent to the method.

[0032] FIG. 22 depicts in accordance with various embodiments of the invention, the concept of linking Capillary Electrophoresis with Mass Spectrometry, alongside the strengths of this technique as they pertain to the detection of BNPi_-32 processing in a plasma matrix.

[0033] FIG. 23 depicts in accordance with various embodiments of the invention, the experimental outline used to define the useable plasma dilution. Conclusion: the 1 : 10 dilution provides a readout of BNPi_-32 degradation over a 12 hour timespan.

[0034] FIG. 24 depicts in accordance with various embodiments of the invention, total ion and extracted ion electropherograms for BNPi_-32, BNP_3-32, BNP_{3 -2}9, BNPi_-30 and BNP_3-30 from a single sample injection plug, showing the ability of the CESI-MS method to resolve the five peptidoforms of BNP, namely BNPi_-32, BNP_3-32, BNP_3-29, BNPi_-30, and BNP_3-30.

[0035] FIG. 25 depicts in accordance with various embodiments of the invention, the time course profile of BNPi_-32, BNP_3-32, BNP_3-29, BNPi_-30 and BNP_3-30 as individual peak areas over a total of 14hrs post-pulse (n=3).

[0036] FIG. 26 depicts in accordance with various embodiments of the invention, the ability of our assay to distinguish BNP cleavage profiles according to different storage conditions over a 12h timespan. Upper right panel: profile shows that plasma stored frozen (- 80°C) and thawed prior to analysis shows an equivalent cleavage profile for BNPi_-32 as plasma stored at 4°C for 24h, while plasma storage at room temperature showed an altered profile. Lower left panel: Cleavage profiles are similar up to 3 freeze-thaw cycles, but adversely affected by 4 or more freeze-thaw cycles. Right panels: Likewise, fresh-frozen plasma analyzed immediately upon thawing (upper right) resulted in a qualitatively dissimilar profile over 12hrs to plasma stored for 24hrs at 4°C (right panels). Conclusion: This assay requires consistency in plasma storage; plasma should be stored at -80°C upon sampling, and thawed immediately prior to analysis.

[0037] FIG. 27 depicts in accordance with various embodiments of the invention, a workflow, inherent to multi-segment injection, designed to incorporate external (day-to-day) and internal (analytical) QC. By incorporating one segment for an internal standard QC to monitor analytical and instrument performance, and a second segment for internal study QC to monitor non-enzymatic degradation, each run can be independently assessed for quality. An additional daily analysis of an external QC of freshly prepared BNPi_-32 in water can further evaluate instrument performance prior to routine clinical analysis.

[0038] FIG. 28 depicts in accordance with various embodiments of the invention, representative results of incorporating internal standard QC and internal study QC metrics as two separate segments for multi-segment injection CESI-MS for BNP cleavage profiling in plasma.

[0039] FIG. 29 depicts in accordance with various embodiments of the invention, differential BNP peptidoform profiles for three patients across a 12hr timeframe. Upper panels show qualitative differences in the combined profiles for BNPi_-32, and its cleavage peptidoforms BNPi_-3o, BNP_3-29, BNP_3-3o, and BNP_3-32, within plasma three different patients. Lower panels separately show differential profiles for BNPi_-32, B P_3-32, BNP_3-30 between three patients.

[0040] FIG. 30 depicts in accordance with various embodiments of the invention, an iteration of our approach in which a 5-point proteolysis profile is produced from plasma within one hour. Upper panel depicts the time breakdown for each experimental phase. The lower left panel shows the representative extracted ion electropherograms, while the lower right panel shows the 5-point profile of the ratio of Β Ρ_3-32:Β Ρι_-32 over 30 minutes.

DETAILED DESCRIPTION OF THE INVENTION

[0041] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and Practice of Pharmacy 22^nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al, Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March 's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3^rd ed., Wiley -Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al, Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0042] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0043] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, kits, systems, articles, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0045] Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application. [0046] As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease, disorder or medical condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, "treatment" may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. Non-limiting examples of treatments or therapeutic treatments include pharmacological therapies (including but not limited to angiotensin receptor blockers, Acetylcholinesterase inhibitors, Aldosterone inhibitors, Beta-blockers, Diuretics) and/or interventional surgical treatments (including but not limited to bypass surgery, valve surgery, left ventricular assist devices).

[0047] In various embodiments, the treatments or kits may be provided as pharmaceutical compositions. In various embodiments, the pharmaceutical compositions may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, enteral, topical or local. "Parenteral" refers to a route of administration that is generally associated with injection, including intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the pharmaceutical compositions can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions. Methods for these administrations are known to one skilled in the art. In certain embodiments, the pharmaceutical compositions are formulated for intravascular, intravenous, or intraarterial administration.

[0048] In various embodiments, the pharmaceutical compositions can contain any pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, nontoxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of excipients include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

[0049] In various embodiments, the pharmaceutical compositions can contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its imaging benefits.

[0050] The pharmaceutical compositions can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0051] The pharmaceutical compositions are made following the conventional techniques of pharmacy involving dry milling, mixing, and blending for powder forms; milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0052] Before administration to patients, formulants may be added to the pharmaceutical composition. A liquid formulation may be preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents or combinations thereof.

[0053] Carbohydrate formulants include sugar or sugar alcohols such as monosaccharides, disaccharides, or polysaccharides, or water soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. In one embodiment, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %.

[0054] Amino acids formulants include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.

[0055] Polymers formulants include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.

[0056] A buffer may also be used in the pharmaceutical compositions to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used including but not limited to citrate, phosphate, succinate, and glutamate buffers or mixtures thereof. In some embodiments, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268, 110.

[0057] After the pharmaceutical composition is prepared, it may be lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing pharmaceutical compositions are known to those of ordinary skill in the art. Just prior to use, the pharmaceutical composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the pharmaceutical composition is administered to subjects using those methods that are known to those skilled in the art.

[0058] The pharmaceutical compositions may be sterilized by conventional, well-known sterilization techniques. The resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The pharmaceutical compositions may contain pharmaceutically-acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and stabilizers (e.g., 1-20% maltose, etc.).

[0059] Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

[0060] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, time, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0062] "Beneficial results" or "desired results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy. As non-limiting examples, "beneficial results" or "desired results" may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a cardiovascular disease, delay or slowing of a cardiovascular disease, and amelioration or palliation of symptoms associated with a cardiovascular disease.

[0063] As used herein, the term "administering," refers to the placement an agent or a treatment as disclosed herein into a subject by a method or route which results in at least partial localization of the agent or treatment at a desired site. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, via inhalation, oral, anal, intra-anal, peri-anal, transmucosal, transdermal, parenteral, enteral, topical or local. "Parenteral" refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the pharmaceutical compositions can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions. In accordance with the present invention, "administering" can be self-administering. For example, it is considered as "administering" that a subject consumes a composition as disclosed herein.

[0064] The term "Brain natriuretic peptide" or "B type natriuretic peptide" or "BNP" or "B P 1-32" as used herein refers to the mature 32-amino acid B type natriuretic peptide molecule.

[0065] A "cardiovascular disease," as used herein, refers to a disorder of the heart and blood vessels, and includes disorders of the arteries, veins, arterioles, venules, and capillaries. Non-limiting examples of cardiovascular diseases diagnosed by a method described herein can include congestive heart failure (HF), coronary artery disease (CAD), arrhythmia, pericarditis, and acute myocardial infarction (MI). Non-limiting examples of cardiovascular disease include: coronary artery disease, coronary heart disease, ischemic heart disease (IHD), cardiomyopathy, stroke, hypertensive heart disease, heart failure, pulmonary heart disease, ischemic syndrome, coronary microvascular disease, cardiac dysrhythmias, rheumatic heart disease (RHD), aortic aneurysms, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, inflammatory heart disease, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, cerebrovascular disease, and peripheral artery disease (PAD).

[0066] As used herein, the term "heart failure" refers to the pathophysiological state in which the heart is unable to pump blood at a rate commensurate with the requirements of the metabolizing tissues or can do so only from an elevated filling pressure.

[0067] "Diagnostic" means identifying the presence or nature of a pathologic condition and includes identifying patients who are at risk of developing a specific disease or disorder. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0068] By "at risk of is intended to mean at increased risk of, compared to a normal subject, or compared to a control group, e.g. a patient population. Thus a subject carrying a particular marker may have an increased risk for a specific disease or disorder, and be identified as needing further testing. "Increased risk" or "elevated risk" mean any statistically significant increase in the probability, e.g., that the subject has the disorder. The risk is preferably increased by at least 10%, more preferably at least 20%, and even more preferably at least 50% over the control group with which the comparison is being made.

[0069] The term "statistically significant" or "significantly" refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p- value.

[0070] The terms "detection", "detecting" and the like, may be used in the context of detecting cleavage products of the natriuretic peptides, or of detecting a disease or disorder (e.g. when positive assay results are obtained). In the latter context, "detecting" and "diagnosing" are considered synonymous.

[0071] The term "diagnosis," or "dx," refers to the identification of the nature and cause of a certain phenomenon. As used herein, a diagnosis typically refers to a medical diagnosis, which is the process of determining which disease or condition explains a symptoms and signs. A diagnostic procedure, often a diagnostic test or assay, can be used to provide a diagnosis. A diagnosis can comprise detecting the presence of a disease or disorder or the risk of getting a disease or disorder

[0072] The term "prognosis," or "px," as used herein refers to predicting the likely outcome of a current standing. For example, a prognosis can include the expected duration and course of a disease or disorder, such as progressive decline or expected recovery.

[0073] The term "theranosis," or "tx" as used herein refers to a diagnosis or prognosis used in the context of a medical treatment. For example, theranostics can include diagnostic testing used for selecting appropriate and optimal therapies (or the inverse) based on the context of genetic content or other molecular or cellular analysis. Theranostics includes pharmacogenomics, personalized and precision medicine.

[0074] "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)'₂ fragments. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHi, CH₂ and CH₃, but does not include the heavy chain variable region.

[0075] Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

[0076] The terms "subject", "patient" or "individual" generally refer to a human, although the methods of the invention are not limited to humans, and should be useful in other animals (e.g. birds, reptiles, amphibians, mammals), particularly in mammals, since albumin is homologous among species. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, "patient", "individual" and "subject" are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein can be used to treat domesticated animals and/or pets. [0077] "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0078] The terms "cleavage products" and "cleavage peptidoforms" have the same meaning and are used interchangeably herein, whereas the term "peptidoforms" encompasses the parent Β Ρι_-32 peptidoform as well as its cleavage peptidoforms.

[0079] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cardiovascular disease) or one or more complications related to the condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to the condition or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular condition can be a subject suspected of having that condition, diagnosed as having that condition, already treated or being treated for that condition, not treated for that condition, or at risk of developing that condition.

[0080] "Sample" is used herein in its broadest sense. The term "biological sample" as used herein denotes a sample taken or isolated from a biological organism. A sample or biological sample may comprise a bodily fluid including blood, serum, plasma, tears, aqueous and vitreous humor, spinal fluid; a soluble fraction of a cell or tissue preparation, or media in which cells were grown; or membrane isolated or extracted from a cell or tissue; polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; fragments and derivatives thereof. Non-limiting examples of samples or biological samples include cheek swab; mucus; whole blood, blood, serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; and tissue sample etc. The term also includes a mixture of the above-mentioned samples or biological samples. The term "sample" also includes untreated or pretreated (or pre- processed) biological samples. In some embodiments, a sample or biological sample can comprise one or more cells from the subject. Subject samples or biological samples usually comprise derivatives of blood products, including blood, plasma and serum.

[0081] Sample collection procedures and devices known in the art are suitable for use with various embodiment of the present invention. Examples of sample collection procedures and devices include but are not limited to: phlebotomy tubes (e.g., a vacutainer blood/specimen collection device for collection and/or storage of the blood/specimen), dried blood spots, Microvette CB300 Capillary Collection Device (Sarstedt), HemaXis blood collection devices (microfluidic technology, Hemaxis), Volumetric Absorptive Microsampling ( such as CE-IVD Mitra microsampling device for accurate dried blood sampling (Neoteryx), HemaSpot™-HF Blood Collection Device.

[0082] The terms "proteases" and "peptidases" are used interchangeably herein to mean enzymes that breakdown proteins and peptides.

[0083] An array of B P precursors and cleavage products exist in blood in an equilibrium established by the rate at which precursors are produced and secreted, as well as the rate at which substrates are cleaved. The components of this equilibrium bind to receptors with unequal affinities, and the strength of the signaling cascades that they induce results from the collective sum of these unequal contributors. Assays that measure BNP are incapable of correctly measuring diverse BNP-like products and cannot analytically describe this equilibrium. As soon as plasma is sampled from a patient, the input of new BNP substrates ceases, but the activity of catabolic processes does not. This artificially skews the equilibrium. Secondly, analytical methods for BNP are either incapable of distinguishing between these peptidoforms, insufficiently sensitive, or both. The methods described herein bypasse the sensitivity problem by spiking in excess BNP into plasma samples. The rate at which new BNP cleavage products appear over time is thus indicative of plasma peptidase activities. These activities are a fundamental component in establishing the aforementioned equilibrium, and therefore more directly related to maladaptive signaling in cardiovascular disease.

[0084] There is a controversy around which assay to use to monitor/assess heart failure and hemodynamic status of an individual. There are two clinical assays proNT and BNP. ProNT is a large fragment generated from the pre-hormone when BNP (32 amino acids), which is cleaved by a series of enzymes (neutral endopeptidase, dipeptidylpeptidase IV, and insulin degrading enzyme). BNP has been reported to be further cleaved in plasma. The inventors have built an assay that can provide a reliable measure of enzymatic activity which should better reflect the biological consequences of B P-mediated signaling in cardiovascular disease. We can additionally monitor formation of two or more fragments of BNP. There are 2 major problems with existing methods: 1) BNP and NT-proBNP assays were developed and used in clinical lab for heart failure diagnosis. However, both of them are all antibody-based ELISA, and those antibodies cannot differentiate BNP, proBNP and their variants. So the measurement itself is not accurate. 2) Based on the inventors' studies, after blood collection, the enzymes are still active and keeping on digesting BNP. Unlike in vivo, the generation of BNP is stopped and the dynamic balance of BNP and its peptidoforms are changed before the test, so the current assay cannot tell the real status of patient by measuring unstable targets. Currently, there are no existing solutions to these problems. The inventors have identified unique peptides to the N- and C-terminus of BNP, such as BNP3-32, BNP 3- 29 etc. This process maybe similar for the other natriuretic peptides (CNP and ANP) and similar enzymatic assays could be produced.

[0085] Our goal was to develop a mass spectrometry (MS) based method capable of identifying and profiling BNP cleavage peptidoforms formed in plasma samples, with a view to informing the degradation of intact BNP in plasma samples. In developing a technique that can account for BNP cleavage peptidoforms, we aimed to help clarify the extent to which BNP is degraded by endogenous factors in plasma with the ultimate goal of providing more accurate diagnostic and prognostic parameters for heart failure.

[0086] We identified several requirements and impediments to the development of such a method. First, in order to avoid skewing the proteolytic profile by disfavoring or outright elimination of potential peptidoforms during sample preparation, the method must almost certainly involve intact protein analysis. Second, since clinically useful BNP concentrations range between 100-400pg^L, we anticipated that the direct measurement of endogenous BNP cleavage peptidoforms would require analytical sensitivity into the low to sub-ng^L range. Third, a method for endogenous BNP proteolytic profiling would have to achieve sufficient sensitivity and simultaneously overcome the inherently broad dynamic range of plasma, which often involves an enrichment step. Immunoprecipitation-based sample enrichment strategies represent the obvious approach, but these can also skew proteolytic profiles by disfavoring peptidoforms with altered epitopes. Furthermore, enrichment strategies and similarly complex sample preparation steps lengthen protocols and increase variance, and are unlikely to ultimately be adopted for routine clinical analyses.

[0087] In this study, we present an alternative approach for BNP proteolytic profiling based on using neutral-coated capillary electrophoresis with electrospray ionization in a single unit that is directly coupled to a mass spectrometry (CESI-MS), and that can measure individual BNP cleavage peptidoforms as they are generated over time in minimally processed plasma. In our approach, standard exogenous BNPi_-32 is pulsed into a plasma sample, where endogenous peptidases cleave BNPi_-32 into its peptidoforms, which are then detected and profiled over time. This reaction can proceed in a CE sample vial that can be sampled for analysis at any desired time interval. By integrating multisegment injection (MSI), where multiple samples are sequentially introduced into a single capillary for simultaneous analysis, our method allows for the parallel analysis of multiple plasma samples where successive CESI-MS runs providing a time course for BNP proteolytic profiling. Similarly, MSI can be used to produce a multi-point BNP proteolytic profile from one plasma sample across the protracted timeframe of a single CESI-MS run.

[0088] The methods described herein comprise adding recombinant intact BNP into patient serum/plasma, and utilize mass spectrometry to analyze the proteolysis profile or proteolytic profile of patients to establish a risk assessment method for cardiovascular diseases.

[0089] Provided herein is a method for assessing risk of cardiovascular disease in a subject in need thereof. The method includes obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; determining the protease activity over time comprising quantifying cleavage products of the one or more natriuretic peptide over of time, wherein an increase in the one or more cleavage products over time is indicative of increased protease activity; and assessing the risk of cardiovascular disease. In one embodiment, the subject has increased risk of cardiovascular disease if the protease activity is increased over time relative to the reference sample. In another embodiment, the subject has a decreased risk of cardiovascular disease if the protease activity is decreased over time relative to the reference sample.

[0090] In various embodiments, the present invention provides a method for determining the risk of developing cardiovascular disease in a subject, comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring an amount of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein an increase in the amount of one or more cleavage products over the period of time is indicative of increased protease activity; determining that the subject has an increased risk of developing cardiovascular disease if the protease activity is increased over the period of time relative to a reference value; determining that the subject has a decreased risk of developing cardiovascular disease if the protease activity is decreased over the period of time relative to the reference value; and selecting a treatment for the subject if the increased risk of developing cardiovascular disease is determined. In some embodiments, the method further comprises measuring an amount of the one or more natriuretic peptides over a period of time.

[0091] Provided herein is a method for assessing risk of cardiovascular disease in a subject in need thereof. The method includes obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; and determining the rate at which the cleavage products of the one or more natriuretic peptides appear over time. In various embodiments, the rate at which the cleavage products appear over time is indicative of the peptidase activity in the sample. In one embodiment, an increase in peptidase activity relative to reference value is indicative of increased likelihood of cardiovascular disease.

[0092] In various embodiments, the present invention provides a method for determining the risk of developing cardiovascular disease in a subject, comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring a rate at which one or more cleavage products of the one or more natriuretic peptides is detected over a period of time, wherein an increase in the rate at which the one or more cleavage products is detected over the period of time is indicative of increased protease activity; determining that the subject has an increased risk of developing cardiovascular disease if the protease activity is increased over the period of time relative to a reference value; determining that the subject has a decreased risk of developing cardiovascular disease if the protease activity is decreased over the period of time relative to the reference value; and selecting a treatment for the subject if the increased risk of developing cardiovascular disease is determined.

[0093] In various embodiments, the present invention provides a method for treating a subject at risk of developing cardiovascular disease, comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring an amount of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein an increase in the amount of one or more cleavage products over the period of time is indicative of increased protease activity; determining that the subject has an increased risk of developing cardiovascular disease if the protease activity is increased over the period of time relative to a reference value; determining that the subject has a decreased risk of developing cardiovascular disease if the protease activity is decreased over the period of time relative to the reference value; and treating the subject having increased protease activity to reduce the risk of developing cardiovascular disease.

[0094] In various embodiments, the present invention provides a method of obtaining a proteolytic profile of one or more cleavage products of one or more natriuretic peptides for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over a period of time to obtain the proteolytic profile of the subject.

[0095] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining one or more samples from the subject, each sample comprising one or more proteases; adding a quantity of one or more natriuretic peptides to each sample; measuring or quantifying the amount of the one or more natriuretic peptides in each sample over a period of time; and measuring or quantifying an amount of one or more cleavage products of the one or more natriuretic peptides in each sample over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the measuring or quantifying of each sample is performed simultaneously. In some embodiments, the measuring or quantifying of each sample is performed sequentially. In some embodiments, the one or more samples is 1-2, 1-3, 1-4, 1 to 5, 2-3, 2-4, 2-5, 3-4, 3-5 or 4-5.

[0096] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring or quantifying the amount of the one or more natriuretic peptides over a period of time; and measuring or quantifying an amount of one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In another embodiment, wherein a sample is one sample.

[0097] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises comparing the proteolytic profile from the subject to a proteolytic profile from a reference sample. In some embodiments, the method further comprises making an assessment of the subject based on the comparison, wherein the assessment is a diagnosis of a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment. In some embodiments, the method further comprises treating the subject for the cardiovascular disease based on the assessment.

[0098] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises comparing the proteolytic profile from the subject to a proteolytic profile from a reference sample. In some embodiments, the method further comprises making an assessment of the subject based on the comparison, wherein the assessment is a prognosis of developing a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment.

[0099] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises comparing the proteolytic profile from the subject to a proteolytic profile from a reference sample, wherein a difference between the proteolytic profile of the subject and the proteolytic profile of the reference sample is indicative of a cardiovascular disease.

[00100] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises comparing the proteolytic profile from the subject to a proteolytic profile from a reference sample, wherein a difference between the proteolytic profile of the subject and the proteolytic profile of the reference sample is an assessment of the subject, wherein the assessment is a diagnosis of a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment. In some embodiments, the method further comprises treating the subject for the cardiovascular disease based on the assessment.

[00101] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises comparing the proteolytic profile from the subject to a proteolytic profile from a reference sample, wherein a difference between the proteolytic profile of the subject and the proteolytic profile of the reference sample is an assessment of the subject, wherein the assessment is a prognosis of developing a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment.

[00102] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises making an assessment of the subject based on the proteolytic profile, wherein the assessment is a diagnosis of a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment. In some embodiments, the method further comprises making an assessment of the subject based on the proteolytic profile, wherein the assessment is a diagnosis of a cardiovascular disease; and treating the subject based on the assessment.

[00103] In various embodiments, the present invention provides a method of obtaining a proteolytic profile for a subject, the method comprising: obtaining a sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the sample; measuring the amount of the one or more natriuretic peptides over a period of time; and measuring an amount of the one or more cleavage products of the one or more natriuretic peptides over the period of time to obtain the proteolytic profile of the subject. In some embodiments, the method further comprises making an assessment of the subject based on the proteolytic profile, wherein the assessment is a prognosis of developing a cardiovascular disease. In some embodiments, the method further comprises treating the subject based on the assessment. In some embodiments, the method further comprises making an assessment of the subject based on the proteolytic profile, wherein the assessment is a prognosis of developing a cardiovascular disease; and treating the subject based on the assessment.

[00104] In various embodiments, the present invention provides a method for assessing the efficacy of a treatment, comprising: comparing a proteolytic profile from a subject to a proteolytic profile from a reference sample, wherein a change in the proteolytic profile from the subject relative to the proteolytic profile from the reference sample is indicative of the efficacy of the treatment.

[00105] In various embodiments, the present invention provides a method for assessing the efficacy of a treatment, comprising: comparing the one or more cleavage products of the one or more natriuretic peptides from a subject to the one or more cleavage products of the one or more natriuretic peptides from a reference sample, wherein a change in the one or more cleavage products from the subject relative to the one or more cleavage products from the reference sample is indicative of the efficacy of the treatment.

[00106] In some embodiments of the present invention, the reference sample is obtained from a control subject, wherein the control subject does not have a cardiovascular disease. In some embodiments of the present invention, the reference sample is obtained from the subject before the subject is treated for a cardiovascular disease. In some embodiments of the present invention, the reference sample is from a subject that has been treated for a cardiovascular disease. In some embodiments of the present invention, the reference sample is obtained from the subject at an earlier time point.

[00107] In various embodiments the present invention provides a method, for determining the risk of developing cardiovascular disease in a subject, comprising: obtaining a biological sample comprising one or more proteases from the subject; adding a quantity of one or more natriuretic peptides to the biological sample; and detecting the presence of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein the presence of one or more cleavage products is indicative of an increased risk of the subject developing cardiovascular disease.

[00108] In various embodiments, assessing the risk of cardiovascular disease in a subject is determining the likelihood of a subject developing cardiovascular diseases. In exemplary embodiments, the cardiovascular disease is heart failure, arterial fibrillation or combination thereof. Specifically, in various embodiments, the invention may provide prognostic or diagnostic information pertaining to categorization of heart failure, for example classification between heart failure with reduced ejection fraction or heart failure with preserved ejection fraction.

[00109] In some embodiments, the natriuretic peptides are any one or more of Brain natriuretic peptide (B P), Atrial natriuretic peptide (A P), C-type natriuretic peptide (CNP) or combinations thereof. In some embodiments, the natriuretic peptide is Brain natriuretic peptide (BNP).

[00110] In exemplary embodiments, the proteases are circulating proteases. In exemplary embodiments, the proteases are any one or more of neutral endopeptidase, dipeptidylpeptidase IV, insulin degrading enzyme or combination thereof. [00111] In some embodiments, the sample is a biological sample. In some embodiments the sample is plasma, blood, or serum. In some embodiments, the sample is plasma. In some embodiments, the biological sample is plasma, blood, or serum. In some embodiments, the biological sample is plasma.

[00112] In exemplary embodiments, the quantity of one or more natriuretic peptides added to the sample is any one or more of about lOng^L, 50ng^L, 75ng^L, lOOng^L, 125ng^L, 150ng^L, 175ng^L, 200ng^L, 225ng^L, 250ng^L, 275ng^L, 300ng^L, 350ng^L, 375ng^L, 400ng^L, 450ng^L, 475ng^L, 500ng^L or combinations thereof. In some embodiments, the quantity of one or more natriuretic peptides added to the sample is any one or more of about 50ng^L, 75ng^L, lOOng^L, 125ng^L, 150ng^L, 175ng^L, 200ng^L, 225ng^L, 250ng/μL or combinations thereof. In some embodiments, the natriuretic peptide is B P and is added in amounts described herein. The optimum amount of the natriuretic peptide to be added to the sample will be apparent to a person of skill in the art.

[00113] In exemplary embodiments, the time period over which the protease activity is determined is about 1 hour in any one or more of 1, 5, 10, 15, 20, 25 or 30 min intervals, about 2 hours in any one or more of 1, 5, 10, 15, 20, 25, 30, 40, 50 or 60 min intervals, about 3 hours in any one or more of 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75 or 90 min intervals, about 4 hours in any one or more of 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 100, 110 or 120 min intervals or combinations thereof. The optimum time period will be apparent to a person of skill in the art. In some embodiments, the time period is less than 1 hour. In some embodiments, the time period is less than or equal to 1 hour. In some embodiments, the time period is about 1 hour. In some embodiments, the time period is about 2 hours. In some embodiments, the time period is about 14 hours. In some embodiments, the time period is about 15 hours. In some embodiments, the time period is up to 1 hour. In some embodiments, the time period is up to 14 hours. In some embodiments the time period is greater than 1 hour. In some embodiments, the time period is greater than 14 hours. In some embodiments, the time period is 0.5 hours to 24 hours. In some embodiments, the time period is 0.5 hours to 20 hours. In some embodiments, the time period is 0.5 hours to 15 hours.

[00114] In some embodiments, the length of the cleavage products of the natriuretic peptides is any one or more of 28-31, 25-30, 20-25, 25-32, 15-20, 10-15, 5-10, 10-20 or 20- 30 amino acids long. [00115] In some embodiments, the natriuretic peptide is BNP 1-32 and the cleavage products comprise any one or more of BNP 3-32, BNP 3-29, BNP 3-30, BNP 1-30, BNP 1- 29, BNP 1-28, BNP 2-31, BNP 4-30, BNP 4-29, BNP 4-27, BNP 5-32, BNP 5-31, BNP 5-29, BNP 4-32, BNP 4-31, or combinations thereof, wherein each range indicates the amino acid position of mature BNP. For example, the full length mature BNP is 32 amino acids long and has amino acids 1-32. "BNP 3-29" refers to the proteolytic fragment of BNP that has amino acids 3-29 of the full length mature BNP. Similarly, "BNP 5-32" refers to the proteolytic fragment of BNP that has amino acids 5-32 of the full length mature BNP.

[00116] In some embodiments, the natriuretic peptide is BNP 1-32 and the cleavage products comprise BNP 3-32, BNP 3-29, BNP 3-30, BNP 1-30, BNP 5-29, BNP 4-29, BNP 1-28, BNP 1-29, BNP 4-31, BNP 4-32 or combinations thereof, wherein each range indicates the amino acid position of mature BNP.

[00117] In some embodiments, the natriuretic peptide is BNP 1-32 and the cleavage products comprise BNP 3-32, BNP 3-29, BNP 3-30, BNP 1-30, or combinations thereof, wherein each range indicates the amino acid position of mature BNP

[00118] In some embodiments, the natriuretic peptide is BNP 1-32 and the cleavage products comprise BNP 3-32, or combinations thereof, wherein each range indicates the amino acid position of mature BNP

[00119] In some embodiments, the cleavage products are not modified. In some embodiments, the cleavage products are modified. In some embodiments, the cleavage products are oxidated at methionine residues. Additional examples of modifications may be found at http://www.unimod.org/modifications_list.php?. In some embodiments, the cleavage products of BNP are not modified. In some embodiments, the cleavage products of BNP are modified. In an exemplary embodiment, the cleavage product of BNP is oxidized at one or more methionine residues.

[00120] In some embodiments, the cleavage products are measured or quantified or detected using any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS), capillary electrophoresis -mass spectrometry (CE-MS), liquid chromatography mass spectrometry (LC-MS), high pressure liquid chromatography mass spectrometry (HPLC-MS), or combinations thereof, in some embodiments, the cleavage products are measured or quantified or detected using capillar}_' electrophoresis-mass spectrometry (CE-MS). In some embodiments, the cleavage products are measured or quantified or detected using capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS). In some embodiments, the cleavage products are measured or quantified or detected using capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS) combined with multi-segment injection (MSI).

[00121] in some embodiments, the cleavage products are measured or quantified or detected using a mass spectrometer and a mass spectrometry method. In some embodiments, the mass spectrometry method comprises any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography mass spectrometry (LC- MS), high pressure liquid chromatography mass spectrometry (HPLC-MS), or combinations thereof. In some embodiments, mass spectrometry data is obtained from the mass spectrometer using the mass spectrometry method.

[00122] In some embodiments, the cleavage products are measured or quantified or detected using a method comprising any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS), capillary electrophoresis -mass spectrometry (CE- MS), liquid chromatography mass spectrometry (LC-MS), high pressure liquid chromatography mass spectrometry (HPLC-MS), or combinations thereof. In some embodiments, the cleavage products are measured or quantified or detected using a method comprising capillary eiectrophoresis-mass spectrometry (CE-MS). In some embodiments, the cleavage products are measured or quantified or detected using a method comprising capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS). In some embodiments, the cleavage products are measured or quantified or detected using a method comprising capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS) combined with multisegment injection (MSI).

[00123] In various embodiments, the cleavage products are measured or quantified or detected using a method comprising capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS) combined with multisegment injection (MSI). In some embodiments, the method further comprises electrokinetic sample injection. In some embodiments, the method further comprises using one or more neutral-coated CESI capillaries. [00124] In some embodiments, the reference value is the protease activity over time in a sample obtained from a healthy subject. In some embodiments, the reference value is the protease activity over time in a sample obtained from a subject that has been treated for cardiovascular disease. In further embodiments, the reference value is the protease activity over time in a sample obtained from the subject at an earlier time point. In some embodiments, the protease activity in a subject having or suspected of having cardiovascular disease is compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the protease activity in a subject having or suspected of having cardiovascular disease is compared to the reference value is increased by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70- fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

Systems and Computers

[00125] In various embodiments, the present invention provides a system for obtaining a proteolytic profile of a subject, comprising: a mass spectrometer configured for acquiring mass spectrometry (MS) data on one or more cleavage products derived from one or more natriuretic peptides in a sample from the subject, wherein the sample comprises one or more natriuretic peptides and one or more proteases; and a computer configured for using the MS data to measure or quantify the amount of one or more cleavage products formed in the sample over a period of time; and for identifying the one or more cleavage products so as to obtain the proteolytic profile of the subject, wherein the mass spectrometer and the computer are connected via a communication link.

[00126] In some embodiments, the computer comprises a memory configured for storing a program; and a processor configured for executing the program, wherein the program comprises instructions for using the MS data to measure or quantify the amount of one or more cleavage products formed in the sample over a period of time; and for identifying the one or more cleavage products so as to obtain the proteolytic profile of the subject.

[00127] A non-transitory computer-readable storage medium, wherein the non-transitory computer-readable storage medium is configured for storing a program, wherein the program is configured for execution by a processor of a computer, and wherein the program comprises instructions for using mass spectrometry (MS) data to measure or quantify the amount of one or more cleavage products derived from one or more natriuretic peptides in a sample from a subject over a period of time; and for identifying the one or more cleavage products, wherein the sample from the subject comprises one or more natriuretic peptides and one or more proteases.

[00128] A computer, comprising: a memory configured for storing a program; and a processor configured for executing the program, wherein the program comprises instructions for processing mass spectrometry (MS) data to measure or quantify the amount of one or more cleavage products derived from one or more natriuretic peptides in a sample from a subject over a period of time; and for identifying the one or more cleavage products, wherein the sample from the subject comprises one or more natriuretic peptides and one or more proteases.

[00129] A computer implemented method, comprising providing a computer, wherein the computer comprises a memory configured for storing a program; and a processor configured for executing the program, wherein the program comprises instructions for processing mass spectrometry (MS) data to measure or quantify the amount of one or more cleavage products derived from one or more natriuretic peptides in a sample from a subject over a period of time; and for identifying the one or more cleavage products, wherein the sample from the subject comprises one or more natriuretic peptides and one or more proteases; inputting MS data into the computer; and operating the computer to process the MS data to measure or quantify the amount of one or more cleavage products derived from one or more natriuretic peptides in the sample over a period of time; and for identifying the one or more cleavage products.

[00130] In accordance with the present invention, a "communication link," as used in this disclosure, means a wired and/or wireless medium that conveys data or information between at least two points. The wired or wireless medium may include, for example, a metallic conductor link, a radio frequency (RF) communication link, an Infrared (IR) communication link, an optical communication link, or the like, without limitation. The RF communication link may include, for example, WiFi, WiMAX, IEEE 802.11, DECT, 0G, 1G, 2G, 3G or 4G cellular standards, Bluetooth, and the like.

[00131] Computers and computing devices typically include a variety of media, which can include computer-readable storage media and/or communications media, in which these two terms are used herein differently from one another as follows. [00132] Computer-readable storage media can be any available storage media that can be accessed by the computer, is typically of a non-transitory nature, and can include both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer-readable storage media can be implemented in connection with any method or technology for storage of information such as computer-readable instructions, program modules, structured data, or unstructured data. Computer-readable storage media can include, but are not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or other tangible and/or non-transitory media which can be used to store desired information. Computer-readable storage media can be accessed by one or more local or remote computing devices, e.g., via access requests, queries or other data retrieval protocols, for a variety of operations with respect to the information stored by the medium.

[00133] On the other hand, communications media typically embody computer-readable instructions, data structures, program modules or other structured or unstructured data in a data signal that can be transitory such as a modulated data signal, e.g., a carrier wave or other transport mechanism, and includes any information delivery or transport media. The term "modulated data signal" or signals refers to a signal that has one or more of its characteristics set or changed in such a manner as to encode information in one or more signals. By way of example, and not limitation, communication media include wired media, such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media.

[00134] Stored on any one or on a combination of computer readable media, the exemplary embodiments of the present disclosure may include software for controlling the devices and subsystems of the exemplary embodiments, for driving the devices and subsystems of the exemplary embodiments, for enabling the devices and subsystems of the exemplary embodiments to interact with a human user, and the like. Such software can include, but is not limited to, device drivers, firmware, operating systems, development tools, applications software, database management software, and the like. Computer code devices of the exemplary embodiments can include any suitable interpretable or executable code mechanism, including but not limited to scripts, interpretable programs, dynamic link libraries (DLLs), Java classes and applets, complete executable programs, and the like. Moreover, processing capabilities may be distributed across multiple processors for better performance, reliability, cost, or other benefits.

[00135] To provide aspects of the present disclosure, embodiments may employ any number of programmable processing devices that execute software or stored instructions. Physical processors and/or machines employed by embodiments of the present disclosure for any processing or evaluation may include one or more networked (Internet, cloud, WAN, LAN, satellite, wired or wireless (RF, cellular, WiFi, Bluetooth, etc.)) or non-networked general purpose computer systems, microprocessors, filed programmable gate arrays (FPGAs), digital signal processors (DSPs), micro-controllers, smart devices (e.g., smart phones), computer tablets, handheld computers, and the like, programmed according to the teachings of the exemplary embodiments. In addition, the devices and subsystems of the exemplary embodiments can be implemented by the preparation of application-specific integrated circuits (ASICs) or by interconnecting an appropriate network of conventional component circuits. Thus, the exemplary embodiments are not limited to any specific combination of hardware circuitry and/or software.

Kits

[00136] In various embodiments, the present invention provides a kit for assessing risk of cardiovascular disease in a subject in need thereof. The kit comprises components to assess the risk of cardiovascular disease in the subject and instructions for use.

[00137] The exact nature of the components configured in the inventive kit depends on its intended purpose. In one embodiment, the kit is configured particularly for human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[00138] Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to assess the risk of cardiovascular disease in a subject. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, pharmaceutical compositions, pharmaceutically acceptable carriers, syringes or other useful paraphernalia as will be readily recognized by those of skill in the art. [00139] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a composition containing a volume of the AAV1-P0-ICE vector. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

[00140] In various embodiments, the present invention provides a kit for identifying a subject at risk of developing a cardiovascular disease, the kit comprising one or more natriuretic peptides. In some embodiments, the kit further comprises instructions for using the kit to identify whether the subject is at risk of developing the cardiovascular disease.

[00141] In various embodiments, the present invention provides a kit for obtaining a proteolytic profile of one or more cleavage products of one or more natriuretic peptides for a subject, the kit comprising one or more natriuretic peptides. In some embodiments, the kit further comprises instructions for using the kit to obtain the proteolytic profile of the subject.

[00142] Some embodiments of the present invention can be defined as any of the following numbered paragraphs:

1. A method, for determining the risk of developing cardiovascular disease in a subject, comprising:

obtaining a biological sample comprising one or more proteases from the subject;

adding a quantity of one or more natriuretic peptides to the biological sample; and detecting the presence of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein the presence of one or more cleavage products is indicative of an increased risk of the subject developing cardiovascular disease. 2. The method of paragraph 1, further comprising selecting one or more treatments for the subject if the increased risk of developing cardiovascular disease is determined.

3. The method of paragraph 1, wherein the natriuretic peptides are any one or more of Brain natriuretic peptide (BNP), Atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) or combinations thereof.

4. The method of paragraph 1, wherein the proteases are any one or more of neutral endopeptidase, dipeptidylpeptidase IV, insulin degrading enzyme or combination thereof.

5. The method of paragraph 1, wherein the sample is plasma, blood, or serum.

6. The method of paragraph 1, wherein the period of time is up to 1 hour.

7. The method of paragraph 1, wherein the period of time is up to 14 hours.

8. The method of paragraph 1, wherein the cleavage products are any one or more of BNP 3- 30, BNP 3-29, BNP 3-32, BNP 1-30 or combinations thereof.

9. The method of paragraph 1, wherein the cleavage products are any one or more of BNP 3- 30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 5-29, BNP 4-29, BNP 1-28, BNP 1-29, BNP 4-31, BNP 4-32 or combinations thereof.

10. The method of paragraph 1, wherein the cleavage products are any one or more of BNP 3-30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 1-29, BNP 1-28, BNP 2-31, BNP 3-30, BNP 4- 30, BNP 4-29, BNP 4-27, BNP 5-32, BNP 5-31, BNP 5-29, BNP 4-32, BNP 4-31, or combinations thereof.

11. The method of paragraph 1, wherein the cleavage products are any one or more of 30-32, 25-30, 20-25, 25-32, 15-20, 10-15, 5-10, 10-20 or 20-30 consecutive amino acids of the natriuretic peptides.

12. The method of any one of paragraphs 8, 9, 10, or 11 wherein the cleavage products are not modified.

13. The method of any one of paragraphs 8, 9, 10, or 11, wherein the cleavage products are modified.

14. The method of paragraph 13, wherein the modification is oxidation at the methionine residue. 15. The method of paragraph 1, wherein the cleavage products are detected using any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI-MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography mass spectrometry (LC-MS), high pressure liquid chromatography mass spectrometry (HPLC- MS), or combinations thereof.

16. The method of paragraph 1, wherein the quantity of one or more natriuretic peptides added to the sample is about any one or more of lOng^L, 50ng^L, 75ng^L, lOOng^L, 125ng^L, 150ng^L, 175ng^L, 200ng^L, 225ng^L, 250ng^L, 275ng^L, 300ng^L, 350ng^L, 375ng^L, 400ng^L, 450ng^L, 475ng^L, 500ng^L or combinations thereof.

17. The method of paragraph 1, wherein the cardiovascular disease is heart failure, arterial fibrillation or combination thereof.

18. The method of paragraph 1, further comprising: comparing the presence of one or more cleavage products of the one or more natriuretic peptides from the subject to the presence of one or more cleavage products of the one or more natriuretic peptides from a reference sample.

19. The method of paragraph 18, further comprising: making an assessment of the subject based on the comparison, wherein the assessment is a determination of the risk of developing cardiovascular disease.

20. The method of paragraph 18, wherein the reference sample is obtained from a healthy subject.

21. The method of paragraph 18, wherein the reference sample is obtained from a subject that has been treated for the cardiovascular disease.

22. The method of paragraph 18, wherein the reference sample is obtained from the subject at an earlier point in time.

23. The method of paragraph 18, wherein the reference sample is obtained from the subject before the subject is treated for the cardiovascular disease.

24. The method of paragraph 1, further comprising detecting the presence of one or more natriuretic peptides over a period of time. EXAMPLES

[00143] The invention is further illustrated by the following examples which are intended to be purely exemplary of the invention, and which should not be construed as limiting the invention in any way. The following examples are illustrative only, and are not intended to limit, in any manner, any of the aspects described herein. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Experimental Methods

[00144] Recombinant human Β Ρι_-32 was purchased from Sigma-Aldrich Cat#B5900, and dissolved in Optima grade water (Fisher Scientific W6500) at 2.5mg/mL. These B Pi_-32 standards were stored at -80°C in ΙΟμΙ. aliquots prior to use. Artificial plasma consisted of 2.25g bovine serum albumin (Recho Ref#: 03117332001) dissolved in 50mL lx PBS pH 7.4 (Quality Biological Cat#: 119-069-131, Lot#: 720744) with 1 tablet of protease inhibitors (Thermo Scientific Cat#: 88266). Human plasma was purchased from Bioreclamation, including human heparin plasma (Cat#: HMPLNAHP, Lot#: BRH181304) and human EDTA plasma (Cat#: HMPLEDTA, Lot#: BRH1120184).

[00145] Plasma sample preparation. All plasma samples were centrifuged through a 0.22μιη spin filter (E&K scientific, Cat#: EK-680850) for 15min at 16100g. Filtered plasma was stored at -80°C in 10μΙ_, aliquots. Plasma aliquots were thawed on ice immediately prior to a CE-MS experiment, and mixed with a designated B Pi_-32 solutions to achieve a final B Pi_-32 concentration of 250ng^L (unless the concentration is otherwise stated).

[00146] Capillary Electrophoresis and Mass Spectrometry. CE System: CE experiments were carried out using a CESI 8000 High Performance Separation-ESI Module (Sciex Separations, Brea, CA). The capillary and sample storage temperatures were maintained at 25°C. The capillary used in this study was the OptiMS Neutral Surface Cartridge (Sciex Separations, Brea, CA). Prior to use, the capillary was first washed by 0.1M hydrochloric acid (Sigma-Aldrich, Cat#258148), then rinsed with background electrolyte (BGE) consisting of 10% acetic acid (Fisher Scientific, Cat#: A38-500), and finally rinsed with deionized water for 30 min at 100 psi and stored overnight filled with water. Before each run, the capillary was rinsed with 0.1M HC1 and flushed with fresh BGE for 10 min at 100 psi. Unless otherwise stated, samples were injected by lOkV voltage for 5 sec and the BGE spacer was added between samples by hydrodynamic injection. A separation voltage of 30 kV was applied across the capillary with a supplemental forward pressure of 1.5 psi.

[00147] Mass Spectrometry. CESI-MS experiments were performed using a Q Exactive+ mass spectrometer (Thermo Fisher Scientific, San Jose, USA). The electrospray voltage used was 1.8kV. Data were acquired with automatic gain control of 3xl0⁶ and a maximum injection time of 100msec. The scan range was set to 200-1200m/z. The MS resolution was set to 70K for the full MS¹ scans, respectively, and the default charge was 4.

[00148] Data Analysis. We performed peptide mapping analysis for the identification and confirmation of B P peptidoforms using Biopharmfinder 1.0 SP1 software (Thermofisher Scientific, San Jose, USA). Accurate MS¹ quantitation of identified peptidoforms was accomplished using Tracefinder 3.1 (Thermofisher Scientific, San Jose, USA).

[00149] This study provides a mass spectrometry-based method capable of reproducibly analyzing Β Ρι_-32 proteolysis profile in a plasma matrix with an overarching longer term vision of providing a deeper understanding of heart failure. In keeping with this goal, we identified the following constraints that our method would have to overcome. First, because a bottom-up proteomics approach (Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M. C; Yates, J. R., 3rd, Protein analysis by shotgun/bottom-up proteomics. Chem Rev 2013, 113 (4), 2343- 94) could directly impact the diversity of the cleavage peptidoforms we seek to detect, our method necessarily must be top-down analysis (Cai, W.; Tucholski, T. M.; Gregorich, Z. R.; Ge, Y., Top-down Proteomics: Technology Advancements and Applications to Heart Diseases. Expert Rev Proteomics 2016, 13 (8), 717-30) of intact proteins. Second, the concentration of endogenous BNP peptidoforms in plasma is estimated in the sub-pg/μΕ range (Maisel, A., B-type natriuretic peptide levels: diagnostic and prognostic in congestive heart failure: what's next? Circulation 2002, 105 (20), 2328-31). In our initial screening experiments, this concentration was within the reach of nano flow LC-MS for the analysis of standard BNP_1-32 reconstituted in a clean aqueous matrix. Unfortunately, achieving this level of sensitivity with sufficient accuracy and reproducibility in plasma samples requires enrichment strategies as well as sample cleanup, and/or fractionation. Finally, while catabolic processes in plasma remain active, sources for secretion of newly synthesized BNPi_-32 are absent in sampled blood. Therefore, analytical accuracy and reproducibility for endogenous BNP peptidoforms may be secondary to the disruption inherent to sample extraction.

[00150] Our approach aims to simultaneously circumvent these constraints by applying a pulse of exogenous standard BNPi-32, followed by direct sampling and analysis of BNP peptidoforms resulting from those intact catabolic processes in a plasma sample. We based our method on a CESI-MS platform for several key reasons: First, BNP is a small protein with a pi of 11, thus carrying a charge across a wide pH range (Table 1). This basic characteristic inherent to BNP and its fragment peptidoforms endows it with an electrophoretic mobility that exceeds a majority of other plasma proteins, which helps minimize interfering signals and plasma matrix effect. Second, the low sample consumption of a given CE run provides an opportunity for multiple successive sampling at different time points from a single vial. Third, CE methods can be built to include MSI, where multiple sample injections separated by short background electrolyte spacers can be simultaneously run and analyzed. The increase in throughput afforded by MSI is an especially attractive feature where the analysis of multiple time points, potential enzyme kinetics, and/or larger clinical cohorts are concerned.

[00151] Our experimental strategy involved the following approach: 1. Establish CESI-MS conditions for the analysis of standard ΒΝΡι-32· 2. Transfer these conditions to the analysis of standard BNPi.3₂ pulsed into plasma. 3. Optimize the method for the simultaneous detection of cleavage peptidoforms in plasma. 4. Explore the application of CESI-MS in one-hour BNP proteolytic profile.

[00152] CESI-MS for standard BNP analysis. Eleven putative BNP cleavage peptidoforms have previously been described in the literature (Niederkofler, E. E.; Kiernan, U. A.; O'Rear, J.; Menon, S.; Saghir, S.; Protter, A. A.; Nelson, R. W.; Schellenberger, U., Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure. Circ Heart Fail 2008, 1 (4), 258-64). These are derived from at least three known BNP-specific proteolytic enzymes; Neutral endopeptidase (NEP), dipeptidylpeptidase IV (DPPIV), and insulin degrading enzyme (IDE) (Volpe, M.; Rubattu, S.; Burnett, J., Jr., Natriuretic peptides in cardiovascular diseases: current use and perspectives. Eur Heart J 2014, 35 (7), 419-25). Table 1 summarizes the theoretical isoelectric points of these peptidoforms in contrast with those of the BNP proteolytic enzymes. Given that our goal was direct sampling from plasma, where the pH falls between the theoretical pi of our panel of potential target analytes and that of the proteolytic enzymes, we built our CE method with electrokinetic sample injection. The use of electrokinetic injection in a neutral pH sample endows our method with the ability to selectively introduce high pi analytes which include all potential BNP peptidoforms, while excluding the lower pi catabolic enzymes responsible for their generation. This technique also simultaneously serves as an endogenous cleanup step that decreases dynamic range and sample plug complexity.

[00153] Table 1. Theoretical pi values for BNP peptidoforms and known plasma proteolytic enzymes wuth uniprot accession number.

[00154] Capillary electrophoresis was performed using commercially available neutral CESI capillaries (Sun, L.; Knierman, M. D.; Zhu, G.; Dovichi, N. I., Fast top-down intact protein characterization with capillary zone electrophoresis-electrospray ionization tandem mass spectrometry. Anal Chem 2013, 85 (12), 5989-95; Neuberger, S.; Rafai, A.; Neususs, C, Screening of Small Intact Proteins by Capillary Electrophoresis Electrospray Ionization- Mass Spectrometry (CE-ESI-MS). Methods Mol Biol 2016, 1466, 43-56) that prevent adsorption and interaction of basic BNP peptidoforms and of intact cationic plasma proteins with the anionic silanol groups that constitute the inner surface of uncoated fused silica capillaries. We established baseline parameters by developing a method for CESI-MS analysis of standard BNPi-32 dissolved in water. This sample was electrokinetically (Hirokawa, T.; Okamoto, H.; Gas, B., High-sensitive capillary zone electrophoresis analysis by electrokinetic injection with transient isotachophoretic preconcentration: electrokinetic supercharging. Electrophoresis 2003, 24 (3), 498-504) injected at 5kV for 10 seconds into a capillary filled with a BGE of 10% acetic acid, and a separation voltage of 30kV supplemented with a forward pressure of 0.5psi was applied throughout the run. We tested the performance of our CESI-MS method by simultaneously analyzing peak migration times (Table 2) and peak areas (Table 3) for four successive runs consisting of five MSI segments each (FIG. 14). These experiments resulted in an average percent of coefficient of variance (%CV) between runs of 7% (N=4), and an average %CV between segments within a run of 21.6%) (N=5). The overall average %>CV for the combination of all segments and all runs was 20%). We found the migration time of the multiply injected peaks, the ESI spray, and the MS signal throughout the CE run to be stable and reproducible.

[00155] Table 2. Intra-run and inter-run migration time of B Pi-32 of CESI-MS with 5 MSI

Migration time (min)

MSI MSI MSI MSI MSI

Run

1 2 3 4 5

1 4.02 6.18 8.39 10.61 12.82

2 4.00 6.20 8.52 10.68 12.92

3 4.06 6.23 8.45 10.67 12.86

4 4.08 6.32 8.56 10.73 12.97

Std dev

/· j. 0.04 0.06 0.08 0.05 0.07 inter-run)

[00156] Table 3. Intra-run and inter-run peak area of B Pi_-32 by CESI-MS with MSI.

Peak Area %CV %CV

(intra- (all runs

Run MSI 1 MSI 3 MSI 4 MSI 5 run) combined)

1 3.4E+08 3.4E+08 3.3E+08 2.9E+08 2.9E+08 8%

2 3.7E+08 3.5E+08 3.6E+08 3.4E+08 3.1E+08 7%

20% 3 3.1E+08 3.3E+08 3.3E+08 3.0E+08 2.8E+08 6%

4 2.2E+08 2.0E+08 2.2E+08 1.9E+08 1.8E+08 7%

%CV

22% 23% 20% 22% 21%

(inter-run) [00157] We applied our method to analyze a dilution series of recombinant B Pi_-32 and generated a linear 5-point standard curve in a single run with five MSI sample injections (FIG. 15). The concentration range of B Pi_-32 for this curve was between 50 ng/μΐ. to 250 ng/μΐ., and all concentrations produced well resolved Gaussian peaks. All five concentration points resulted in CVs under 15% (N=3) and accuracies between 89%- 108% with the exception of the lowest concentration (50ng^L) whose accuracy was 130% (Table 4).

[00158] Table 4. Reproducibility and Accuracy of five B Pi_-32 calibration curves constructed as 5 MSI per curve. C

cy*

50 12% 130%

100 13% 101%

150 15% 89%

200 10% 92%

250 13% 108%

*% accuracy is defined as the quantitative

value of each peak from the calibration curve

relative to its theoretical concentration, and is

calculated using the average of replicates at

the same concentration level.

[00159] CESI-MS for BNP proteolytic profiling in plasma. In order to determine the optimal ratio at which enzymatic components in plasma degrade an exogenous pulse of B Pi_-32 while still being able to monitor substrate, we monitored B Pi_-32 in five parallel plasma dilutions that were analyzed simultaneously with MSI. Segments were injected electrokinetically into the capillary in decreasing order of dilution. Aside from the initial sample preparation and dilution, the incubation and successive overnight analyses were performed within and by the CE instrument as part of a sequence protocol. To our knowledge, this experiment represents the first analysis of proteins from a plasma matrix using a neutral- coated CESI-MS with MSI. This experiment demonstrated that overly diluted plasma (beyond 1 :20) did not result in any appreciable B Pi_-32 degradation. Conversely, we could not detect B Pi_-32 beyond 12hrs when plasma dilutions were 1 :5 or less. A ratio of 1 : 10 allowed us to monitor decreasing B Pi_-32 peaks which remained quantifiable for 12 successive injections spanning 12hrs (FIG. 16). [00160] Over the course of our dilution series experiments, we observed the time- dependent appearance of additional BNP peaks as the degradation reaction proceeded, which we subsequently identified as BNP cleavage peptidoforms with the BioPharma Finder 1.0 Mass Informatics platform (ThermoFisher). We ultimately found that successive sampling across 14hrs from an individual reaction vial enabled the quantitative detection and profiling of a total of five BNP peptidoforms enzymatically generated in plasma, namely BNPi-32 and four of its cleavage peptidoforms: BNP3-32, BNP3-29, BNPi-3o, and ΒΝΡ3-30· Interestingly, the profiles with which these peptidoforms are detected suggest that products of one cleavage reaction may serve as substrates for other reactions. Accordingly, we propose that the simultaneous measurement of BNP peptidoforms over time is a measure of the collective enzymatic processes that catabolize BNP in a plasma sample (FIG. 17A- FIG. 17B).

[00161] One-hour proteolytic profile by CESI-MS with MSI. The prospective application of our proteolytic profiling technique for discovery and research cohorts, warrants the quantitative detection and profiling of as many peptidoforms as possible. The disadvantage inherent to a comprehensive characterization of BNPi-32 along with its four BNP cleavage peptidoforms is its requirement for a 14hr protocol to allow sufficient reaction time for the generation of slower-forming cleavage peptidoforms. We propose that incorporating MSI into the method can increase throughput by enabling the simultaneous analysis of multiple different plasma samples within a single MS run, thereby achieving a faster turnaround. Conversely, we also applied MSI orthogonally to sample a single individual reaction vial every 3 minutes for a total of 5 closely spaced time points. This iteration of our method can provide an acute quantitative profile of primary BNP proteolysis products, which we depict as the ratio of BNP3-32:BNPi.32, from plasma in less than one hour, including sample preparation. Although this lhr method under the current conditions can detect other peptidoforms, the short reaction time precludes a reliable signal with sufficient intensity for their quantitative analysis. Alternatively, adjusting the plasma dilution ratio may facilitate the quantitative analysis of slower-forming BNP peptidoforms within this lhr timeframe.

[00162] EDTA and heparin collection tubes are commonplace for plasma sampling in the hospital setting. Buckley et al (Belenky, A.; Smith, A.; Zhang, B.; Lin, S.; Despres, N.; Wu, A. H.; Bluestein, B. L, The effect of class-specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma. Clin Chim Acta 2004, 340 (1-2), 163-72) suggested EDTA collection tubes be employed where BNP analysis from plasma is concerned when using traditional radioimmunoassay-based methods. While our results clearly indicate that endogenous plasma proteases retain their ability to cleave BNPi_-32 after plasma collection, our study was not designed to assess the extent to which protease activities alter the quantitative accuracy of immunoassay-based methods. Neither was this study designed to assess the suitability of various sampling tubes as they pertain to CESI-MS-based profiling of BNP proteolysis. Nevertheless, our assay provides a measure of the outcome of all plasma proteases that degrade BNP, providing a biological readout of the underlying physiology or pathophysiology. Furthermore, as an initial application for our 1-hr detection method, we performed a pilot side-by-side analysis to compare human plasma collected in either Heparin tubes or EDTA tubes (FIG. 18). In one hour, our method clearly describes a difference in the profiles of BNPi_-32:BNP_{3 3}2 between plasma collected in these two common tubes. Among the enzymes known to cleave BNP, both insulin degrading enzyme (IDE) and neutral endopeptidase (NEP) are known to use Zn²⁺ as a cofactor (Shen, Y.; Joachimiak, A.; Rosner, M. R.; Tang, W. J., Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 2006, 443 (7113), 870-4; Oefner, C; Roques, B. P.; Fournie- Zaluski, M. C; Dale, G. E., Structural analysis of neprilysin with various specific and potent inhibitors. Acta Crystallogr D Biol Crystallogr 2004, 60 (Pt 2), 392-6; Oefner, C; Pierau, S.; Schulz, H.; Dale, G. E., Structural studies of a bifunctional inhibitor of neprilysin and DPP- IV. Acta Crystallogr D Biol Crystallogr 2007, 63 (Pt 9), 975-81). IDE can cut the last 3 amino acids from the C-terminal of BNP while NEP can cleavage between the fourth and fifth amino acids from N- terminal in reactions required to produce the primary proteolysis products of BNP (Volpe, M.; Rubattu, S.; Burnett, J., Jr., Natriuretic peptides in cardiovascular diseases: current use and perspectives. Eur Heart J 2014, 35 (7), 419-25). We have previously detected BNP_{3 -}29 in plasma from heparin collection tubes within an hour, but not from EDTA tubes which require substantially longer incubation times. Without being bound by theory, we hypothesize that the activity of IDE may be partially inhibited by EDTA due to its chelation of Zinc ions.

[00163] Both the plasma dilution series and the absence of BNPi_-32 degradation was the product of previously described enzymatic activities in plasma samples, we measured the time-dependent ratio of BNPi_-32:BNP_{3 -3}2 of the BNPi_-32 pulsed into a commercially available human plasma matrix along with the same ratio that is also pulsed into an artificial plasma matrix consisting of albumin dissolved in PBS. In contrast with the human plasma matrix, a small amount of BNP_{3 -3}2 was consistently detected in the artificial plasma group, but the BNPi_-32:BNP_3-32 ratio was stable (FIG. 18). We did not detect any signals for BNP_{3 -2}9, BNPi. 30, or B P_{3 -}3o in artificial plasma across the timespan of this experiment, and without being bound by theory we hypothesize that the residual B P_3-32 peak represents a low-level degradation product. However, these data confirm that the degradation of B Pi_-32 that we detect in human plasma is enzyme mediated.

[00164] In this study, we present a novel approach in which BNP cleavage peptidoforms are profiled using CESI-MS, as they are enzymatically generated over time in minimally processed plasma. In our approach, standard exogenous B Pi_-32 is pulsed into a plasma sample, where endogenous plasma peptidases cleave BNP to produce various product peptidoforms. This reaction can proceed in a temperature-controlled sample vial, and selectively sampled into a capillary using electrokinetic injection at any desired time interval. We present the first use of MSI with CESI-MS using a neutral coated capillary for proteins in plasma. The use of MSI can endow this method with increased throughput via parallel analysis of multiple plasma samples. In this case, each parallel sample would be represented by an individual sequentially injected segment, and successive CESI-MS runs would provide a time course profile for BNP peptidoform formation across an entire CE sequence. Similarly, we applied MSI orthogonally to the same sample, producing a multi-point BNP peptidoform profile from an individual plasma sample across the protracted timeframe of a single CESI- MS run. This second iteration of our method produces a 5-point profile in under an hour, including all sample preparation steps.

[00165] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[00166] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[00167] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[00168] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[00169] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[00170] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[00171] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[00172] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[00173] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.

Claims

adding a quantity of one or more natriuretic peptides to the biological sample; and detecting the presence of one or more cleavage products of the one or more natriuretic peptides over a period of time, wherein the presence of one or more cleavage products is indicative of an increased risk of the subject developing cardiovascular disease.

2. The method of claim 1, further comprising selecting one or more treatments for the subject if the increased risk of developing cardiovascular disease is determined.

3. The method of claim 1, wherein the natriuretic peptides are any one or more of Brain natriuretic peptide (BNP), Atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) or combinations thereof.

4. The method of claim 1, wherein the proteases are any one or more of neutral endopeptidase, dipeptidylpeptidase IV, insulin degrading enzyme or combination thereof.

5. The method of claim 1, wherein the sample is plasma, blood, or serum.

6. The method of claim 1, wherein the period of time is up to 1 hour.

7. The method of claim 1, wherein the period of time is up to 14 hours.

8. The method of claim 1, wherein the cleavage products are any one or more of BNP 3- 30, BNP 3-29, BNP 3-32, BNP 1-30 or combinations thereof.

9. The method of claim 1, wherein the cleavage products are any one or more of BNP 3- 30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 5-29, BNP 4-29, BNP 1-28, BNP 1-29, BNP 4-31, BNP 4-32 or combinations thereof.

10. The method of claim 1, wherein the cleavage products are any one or more of BNP 3- 30, BNP 3-29, BNP 3-32, BNP 1-30, BNP 1-29, BNP 1-28, BNP 2-31, BNP 3-30, BNP 4-30, BNP 4-29, BNP 4-27, BNP 5-32, BNP 5-31, BNP 5-29, BNP 4-32, BNP 4-31, or combinations thereof.

11. The method of claim 1, wherein the cleavage products are any one or more of 30-32, 25-30, 20-25, 25-32, 15-20, 10-15, 5-10, 10-20 or 20-30 consecutive amino acids of the natriuretic peptides.

12. The method of any one of claims 8, 9, 10, or 11 wherein the cleavage products are not modified.

13. The method of any one of claims 8, 9, 10, or 11, wherein the cleavage products are modified.

14. The method of claim 13, wherein the modification is oxidation at the methionine residue.

15. The method of claim 1, wherein the cleavage products are detected using any one or more of capillary electrophoresis/electrospray ionization-mass spectrometry (CESI- MS), capillary el ectrophore sis-mass spectrometry (CE-MS), liquid chromatography mass spectrometry (LC-MS), high pressure liquid chromatography mass spectrometry (HPLC-MS), or combinations thereof.

16. The method of claim 1, wherein the quantity of one or more natriuretic peptides added to the sample is about any one or more of lOng^L, 50ng^L, 75ng^L, lOOng^L, 125ng^L, 150ng^L, 175ng^L, 200ng^L, 225ng^L, 250ng^L, 275ng^L, 300ng^L, 350ng^L, 375ng^L, 400ng^L, 450ng^L, 475ng^L, 500ng^L or combinations thereof.

17. The method of claim 1, wherein the cardiovascular disease is heart failure, arterial fibrillation or combination thereof.

18. The method of claim 1, further comprising: comparing the presence of one or more cleavage products of the one or more natriuretic peptides from the subject to the presence of one or more cleavage products of the one or more natriuretic peptides from a reference sample.

19. The method of claim 18, further comprising: making an assessment of the subject based on the comparison, wherein the assessment is a determination of the risk of developing cardiovascular disease.

20. The method of claim 18, wherein the reference sample is obtained from a healthy subject.

21. The method of claim 18, wherein the reference sample is obtained from a subject that has been treated for the cardiovascular disease.

22. The method of claim 18, wherein the reference sample is obtained from the subject at an earlier point in time.

23. The method of claim 18, wherein the reference sample is obtained from the subject before the subject is treated for the cardiovascular disease.

24. The method of claim 1, further comprising detecting the presence of one or more natriuretic peptides over a period of time.