# Patent

## Title
Methods and compositions for diagnosis or prognosis of cardiovascular disease

## Abstract

The invention provides methods of screening a mammalian subject to determine if the subject is at risk to develop or is suffering from, cardiovascular disease. In one embodiment, the method comprises detecting a measurable feature of at least two biomarkers in an HDL subfraction, or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the subject, wherein the at least two biomarkers are selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof, and comparing the measurable features of the at least two biomarkers from the biological sample to a reference standard, wherein a difference in the measurable features of the at least two biomarkers from the biological sample and the reference standard is indicative of the presence or risk of cardiovascular disease in the subject.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of Application No. 61/079,088, filed Jul. 8, 2008,


STATEMENT OF GOVERNMENT LICENSE RIGHTS
This invention was made with U.S. Government support under NIH grant number HL086798, awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention generally relates to methods, reagents, and kits for diagnosing cardiovascular disease in a subject, and particularly relates to the use of lipoprotein-associated markers to diagnose cardiovascular disease in a subject.
BACKGROUND
Cardiovascular disease is a leading cause of morbidity and mortality, particularly in developed areas such as the United States and Western European countries. The incidence of mortality from cardiovascular disease has significantly decreased in the United States over the past 30 years (see Braunwald, E., N. Engl. J. Med. 337:1360-1369, 1997; Hoyert, D. L., et al., “Deaths: Preliminary Data for 2003” in National Vital Statistics Reports. Hyattsville: National Center for Health Statistics, 2005). Many factors have contributed to this improvement in patient outcome, including the identification of cardiovascular risk factors, the application of medical technologies to treat acute coronary syndrome, and the development of interventions that reduce cardiovascular risk factors. Despite these advances, however, cardiovascular disease remains a leading cause of morbidity and mortality in developed countries (see Hoyert D. L., et al., National Vital Statistics Reports, 2005).
Thus, there is a pressing need to identify markers that may be used for the rapid, accurate and non-invasive diagnosis and/or assessment of the risk of cardiovascular disease, and also to assess the efficacy of interventions designed to slow the initiation and progress of this disorder.
SUMMARY
In accordance with the foregoing, in one aspect, the present invention provides a method of screening a mammalian subject to determine if the subject is at risk to develop, or is suffering from, cardiovascular disease, the method comprising detecting a measurable feature of at least two biomarkers in an HDL subfraction, or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the subject, wherein the at least two biomarkers are selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof, and comparing the measurable features of the at least two biomarkers from the biological sample to a reference standard, wherein a difference in the measurable features of the at least two biomarkers from the biological sample and the reference standard is indicative of the presence or risk of cardiovascular disease in the subject.
In another aspect, the present invention provides a method for diagnosing and/or assessing the risk of CAD in a subject, comprising determining changes in a biomarker profile comprising the relative abundance of at least one, two, three, four, five, ten or more biomarkers in an HDL subfraction or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from a test subject as compared to the predetermined abundance of the at least one, two, three, four, five, ten or more biomarkers from a reference population of apparently healthy subjects, wherein the biomarkers are selected from the biomarkers set forth in TABLE 3, TABLE 4, and TABLE 5.
In another aspect, the present invention provides a method of screening a mammalian subject to determine if a test subject is at risk to develop, is suffering from, or recovering from, cardiovascular disease, the method comprising detecting an alteration in the conformational structure of apoA-I present in the HDL subfraction or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the test subject in comparison to a reference standard, wherein a difference in the conformation of the apoA-I between the biological sample from the test subject and the reference standard is indicative of the presence or risk of cardiovascular disease in the subject.
In another aspect, the present invention provides a method for determining the efficacy of a treatment regimen for treating and/or preventing cardiovascular disease in a subject by monitoring a measurable feature of at least two biomarkers selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof in an HDL subfraction or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the subject during treatment for cardiovascular disease.
In yet another aspect, the present invention provides a kit for determining susceptibility or presence of cardiovascular disease in a mammalian subject based on the detection of at least one measurable feature of at least one biomarker in a biological sample, an HDL subfraction thereof, or a complex containing apoA-I or apoA-II isolated from the biological sample, the kit comprising (i) one or more detection reagents for detecting the at least one measurable feature of the at least one biomarker selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, and (ii) written indicia indicating a positive correlation between the presence of the detected feature of the biomarker and the diagnosis or risk of developing cardiovascular disease.



DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
FIG. 1 presents graphical results demonstrating the receiver operating characteristic (ROC) curve of the prediction of cardiovascular disease (CAD) status based on random permutation analysis, as described in Example 2;
FIG. 2 graphically illustrates the prediction of CAD status by the proteomics CAD risk score “ProtCAD risk score” using a partial least squares discriminate analysis (PLS-DA) model built using a calibration group (as described in Example 2). Using a sensitivity of 80%, the ProtCAD risk score of each subject in the validation group at each permutation was used to predict their CAD status, as described in Example 2;
FIG. 3 graphically illustrates the power of the ProtCAD risk score to discriminate between the CAD samples and healthy control samples based on leave-one-out analysis. The ProtCAD risk score was derived from PLS-DA analysis of MALDI-TOF-MS mass spectra of HDL tryptic digests, using a leave-one-out experiment for all 18 CAD and 20 control subjects, as described in Example 2;
FIG. 4 graphically illustrates the PLS-DA regression vectors (y-axis) of the leave-one-out PLS-DA model that distinguish CAD and control subjects. The x-axis (m/z) represents mass channels of the MALDI-TOF mass spectrum. Positive and negative features on the regression vector indicate an increase and decrease, respectively, of the signals from CAD samples relative to control samples, as described in Example 3;
FIG. 5A graphically illustrates the strong positive feature in the PLS-DA regression vector at m/z 1440.68 identified by LC-MALDI-TOF/TOF MS/MS as corresponding to peptides derived from Lp(a), as described in Example 3;
FIG. 5B graphically illustrates the strong positive feature in the PLS-DA regression vector at m/z 1904.91 identified by LC-MALDI-TOF/TOF MS/MS as corresponding to peptides derived from Lp(a), as described in Example 3;
FIG. 5C illustrates the results of the MASCOT database search of the MS/MS spectrum of the peptide of m/z 1440.68 that identified Lp(a) with a high confidence level (CI100%), as described in Example 3;
FIG. 6A to FIG. 6D graphically illustrate the PLS-DA regression vector features corresponding to apoA-I peptides containing Met112, with pairs of specific informative features at m/z 1411 and 1427 and m/z 2645 and 2661 corresponding to signals detected for M and M+16 respectively, wherein the positive features signify an increase of oxidized form of Met112 peptide, and negative features at m/z 1411 and m/z 2645 indicate a decreased level of the peptide containing unoxidized Met112 in the CAD samples, as described in Example 4;
FIG. 7 graphically illustrates the differential digestion efficiency in CAD HDL as compared to normal HDL, in which multiple features in the regression vector (y-axis) correspond to peptides derived from apoA-I (x-axis) with differential features at the N-terminal (residues 46-59, 60-77) and the C-terminal (residues 207-215) domains, indicating a conformational change in apoA-I in the HDL of CAD subjects, as described in Example 5;
FIG. 8 graphically illustrates the results of principle component analysis (PCA) of the average mass spectra from HDL2 isolated from 3 control and 3 CAD subjects mixed in protein ratios (w/w) of 1:0, 1:3, 1:1, 3:1, and 0:1, digested with trypsin, and subjected to MALDI-TOF-MS (for simplicity only two pairs are shown in FIG. 8), as described in Example 1;
FIG. 9A graphically illustrates the reproducibility of the MALDI-TOF spectra with selected mass channels of Met112 peptides represented on the plot as median normalized intensities, as described in Example 1;
FIG. 9B graphically illustrates the reproducibility of the MALDI-TOF spectra of multiple spots of samples with selected mass channels of Met112 peptides represented on the plot as median normalized intensities, as described in Example 1;
FIG. 9C graphically illustrates the reproducibility of a series of trypsin digestions carried out on the same day followed by MALDI-TOF spectra with selected mass channels of Met112 peptides represented on the plot as median normalized intensities, as described in Example 1;
FIG. 9D graphically illustrates the reproducibility of a series of trypsin digestions carried out on different days followed by MALDI-TOF spectra with selected mass channels of Met112 peptides represented on the plot as median normalized intensities, as described in Example 1;
FIG. 10A graphically illustrates a receiver operating characteristic (ROC) curve constructed using a ProtCAD score based on a PLS-DA model built from a leave-one-out approach, demonstrating high selectivity (true positive rate=y axis) and high specificity (false positive rate=x axis), as described in Example 2; and
FIG. 10B graphically illustrates the odds ratio of the ProCAD score as a function of the false positive rate, demonstrating that at an 80% level of specificity (corresponding to a 90% sensitivity level as shown in FIG. 10A), the odds ratio was approximately 35, as described in Example 2.



DETAILED DESCRIPTION
As used herein, the term “cardiovascular disease” or “CAD,” generally refers to heart and blood vessel diseases, including atherosclerosis, coronary heart disease, cerebrovascular disease, and peripheral vascular disease. Cardiovascular disorders are acute manifestations of CAD and include myocardial infarction, stroke, angina pectoris, transient ischemic attacks, and congestive heart failure. Cardiovascular disease, including atherosclerosis, usually results from the build up of fatty material, inflammatory cells, extracellular matrix, and plaque. Clinical symptoms and signs indicating the presence of CAD include one or more of the following: chest pain and other forms of angina, shortness of breath, sweatiness, Q waves or inverted T waves on an EKG, a high calcium score by CT scan, at least one stenotic lesion on coronary angiography, or heart attack documented by changes in myocardial enzyme levels (e.g., troponin, CK levels).
As used herein, the term “biomarker” is a biological compound, such as a protein or a peptide fragment thereof, including a polypeptide or peptide that may be isolated from or measured in the biological sample, wherein the biomarker is differentially present or absent, or present in a different structure (i.e., post-translationally modified, or in an altered structural conformation) in a sample taken from a subject having established or potentially clinically significant CAD as compared to a comparable sample taken from an apparently normal subject that does not have CAD. A biomarker can be an intact molecule, or it can be a portion thereof or an altered structure thereof, that may be partially functional and recognized, for example, by a specific binding protein or other detection method. A biomarker is considered to be informative for CAD if a measurable feature of the biomarker is associated with the presence of CAD in a subject in comparison to a predetermined value or a reference profile from a control population. Such a measurable feature may include, for example, the presence, absence, or concentration of the biomarker, or a portion thereof, in the biological sample, an altered structure, such as, for example, the presence or amount of a post-translational modification, such as oxidation at one or more positions on the amino acid sequence of the biomarker or, for example, the presence of an altered conformation in comparison to the conformation of the biomarker in normal control subjects, and/or the presence, amount, or altered structure of the biomarker as a part of a profile of more than one biomarker. A measurable aspect of a biomarker is also referred to as a feature. A feature may be a ratio of two or more measurable aspects of biomarkers. A biomarker profile comprises at least two measurable informative features, and may comprise at least three, four, five, 10, 20, 30 or more informative features. The biomarker profile may also comprise at least one measurable aspect of at least one feature relative to at least one internal standard.
As used herein, the term “predetermined value” refers to the amount and/or structure of one or more biomarkers in biological samples obtained from the general population or from a select population of subjects. For example, the select population may be comprised of apparently healthy subjects, such as individuals who have not previously had any sign or symptoms indicating the presence of CAD. In another example, the predetermined value may be comprised of subjects having established CAD. The predetermined value can be a cut-off value or a range. The predetermined value can be established based upon comparative measurements between apparently healthy subjects and subjects with established CAD, as described herein.
As used herein, the term “high density lipoprotein” or “HDL, or a subfraction thereof” includes protein or lipoprotein complexes with a density from about 1.06 to about 1.21 g/mL, or from about 1.06 to 1.10 g/mL, or from about 1.10 to about 1.21 g/mL, or a complex containing apoA-I or apoA-II. HDL may be prepared by density ultracentrifugation, as described in Mendez, A. J., et al., J. Biol. Chem. 266:10104-10111, 1991, from plasma, serum, bodily fluids, or tissue. The HDL3 subfraction in the density range of about 1.110 to about 1.210 g/mL, and the HDL2 subfraction in the density range of about 1.06 to about 1.125 g/mL may be isolated from plasma, serum, bodily fluids, tissue or total HDL by sequential density ultracentrifugation, as described in Mendez, supra. HDL is known to contain two major proteins, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II); therefore, in some embodiments, the term “HDL, or a subfraction thereof” also includes an apoA-I and/or an apoA-II containing protein or lipoprotein complex which may be isolated, for example, by immunoaffinity with anti-apoA-I or anti-apoA-II antibodies.
As used herein, the term “HDL-associated” refers to any biological compounds that float in the density range of HDL (d=about 1.06 to about 1.21 g/mL) and/or molecules present in a complex containing apoA-I and/or apoA-II, including full-length proteins and fragments thereof, including peptides or lipid-protein complexes, such as microparticles, in HDL isolated from any sample, including lesions, blood, urine, cerebral spinal fluid, bronchoalveolar fluid, joint fluid, or tissue or fluid samples.
As used herein, the term “HDL2-associated” refers to any biological compounds that float in the density range of HDL2 (d=about 1.06 to about 1.125 g/mL) and/or molecules present in a complex containing apoA-I and/or apoA-II, including full-length proteins, and fragments thereof, including peptides, or lipid-protein complexes such as microparticles, in HDL isolated from any sample, including lesions, blood, urine, cerebral spinal fluid, bronchoalveolar fluid, joint fluid, or tissue or fluid samples.
As used herein, the term “mass spectrometer” refers to a device able to volatilize/ionize analytes to form gas-phase ions and determine their absolute or relative molecular masses. Suitable forms of volatilization/ionization are matrix-assisted laser desorption ionization (MALDI), electrospray, laser/light, thermal, electrical, atomized/sprayed and the like, or combinations thereof. Suitable forms of mass spectrometry include, but are not limited to, ion trap instruments, quadrupole instruments, electrostatic and magnetic sector instruments, time of flight instruments, time of flight tandem mass spectrometer (TOF MS/MS), Fourier-transform mass spectrometers, and hybrid instruments composed of various combinations of these types of mass analyzers. These instruments may, in turn, be interfaced with a variety of sources that fractionate the samples (for example, liquid chromatography or solid-phase adsorption techniques based on chemical, or biological properties) and that ionize the samples for introduction into the mass spectrometer, including matrix-assisted laser desorption (MALDI), electrospray, or nanospray ionization (ESI) or combinations thereof.
As used herein, the term “affinity detection” or “affinity purified” refers to any method that selectively detects and/or enriches the protein or analyte of interest. This includes methods based on physical properties like charge, amino acid sequence, and hydrophobicity, and can involve many different compounds that have an affinity for the analyte of interest, including, but not limited to, antibodies, resins, RNA, DNA, proteins, hydrophobic materials, charged materials, and dyes.
As used herein, the term “antibody” encompasses antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human) that specifically bind to the biomarkers or portions thereof. Exemplary antibodies include polyclonal, monoclonal, and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact molecule or fragment thereof.
As used herein, the term “antibody fragment” refers to a portion derived from or related to a full length anti-biomarker antibody, generally including the antigen binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab′, F(ab)2, F(ab′)2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Antibody and antibody fragments as used here may be incorporated into other proteins that can be produced by a variety of systems, including, but not limited to, bacteria, viruses, yeast, and mammalian cells.
As used herein, “a subject” includes all mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.
As used herein, the term “percent identity” or “percent identical,” when used in connection with a biomarker used in the practice of the present invention, is defined as the percentage of amino acid residues in a biomarker sequence that are identical with the amino acid sequence of a specified biomarker after aligning the sequences to achieve the maximum percent identity. When making the comparison, no gaps are introduced into the biomarker sequences in order to achieve the best alignment.
Amino acid sequence identity can be determined, for example, in the following manner. The amino acid sequence of a biomarker is used to search a protein sequence database, such as the GenBank database, using the BLASTP program. The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTP are utilized.
As used herein, the term “derivatives” of a biomarker, including proteins and peptide fragments thereof, include an insertion, deletion, or substitution mutant. Preferably, any substitution mutation is conservative in that it minimally disrupts the biochemical properties of the biomarker. Thus, where mutations are introduced to substitute amino acid residues, positively-charged residues (H, K, and R) preferably are substituted with positively-charged residues; negatively-charged residues (D and E) are preferably substituted with negatively-charged residues; neutral polar residues (C, G, N, Q, S, T, and Y) are preferably substituted with neutral polar residues; and neutral non-polar residues (A, F, I, L, M, P, V, and W) are preferably substituted with neutral non-polar residues.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).
In the broadest sense, the naturally occurring amino acids can be divided into groups based upon the chemical characteristic of the side chain of the respective amino acids. By “hydrophobic” amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. By “hydrophilic” amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This grouping of amino acids can be further subclassed as follows. By “uncharged hydrophilic” amino acid is meant either Ser, Thr, Asn, or Gln. By “acidic” amino acid is meant either Glu or Asp. By “basic” amino acid is meant either Lys, Arg, or His.
In the past, studies have been done to identify proteins in the blood of a subject that could be used as markers for cardiovascular disease (see, e.g., Stanley et al., Dis. Markers 20:167-178, 2004). However, this approach has been hampered by the vast number of candidate proteins in blood plasma in concentrations that vary over six orders of magnitude, which complicate the discovery and validation processes (Qian, W. J., et al., Proteomics 5:572-584, 2005). Cholesterol is present in the blood as free and esterified cholesterol within lipoprotein particles, commonly known as chylomicrons, very low density lipoproteins (VLDLs), low density lipoproteins (LDLs), and high density lipoproteins (HDLs). HDL particles vary in size and density due to the differences in the number of apolipoproteins on the surface of the particles and the amount of cholesterol esters in the core of HDL (see Asztaloe et al., Am. J. Cardiol. 91:12 E-17E, 2003). HDL is composed of two principal subfractions based on density: HDL2 and the denser HDL3.
Elevated LDL cholesterol and total cholesterol are directly related to an increased risk of cardiovascular disease. See Anderson et al., “Cholesterol and Mortality: 30 years of Follow Up from the Framingham Study,” JAMA 257:2176-90, 1987. In contrast, it has been established that the risk of cardiovascular disease is inversely proportional to plasma levels of HDL and the major HDL apolipoprotein, apoA-I (Gordon, D. J., et al., N. Engl. J. Med. 321:1311-1316, 1989). Studies have shown that high HDL levels are associated with longevity (Barzilai, N., et al., JAMA 290:2030-2040, 2003). Consistent with these findings, an abnormally low HDL level is a well-accepted risk factor for the development of clinically significant atherosclerosis (particularly common in men with premature atherosclerosis (Gordon, D. J., et al., N. Engl. J. Med. 321:1311-1316, 1989; Wilson, P. W., et al., Arteriosclerosis 8:737-741, 1988)). The mechanism by which HDL renders its protective effect against atherosclerosis is the subject of continued debate. Some studies have implicated that HDL may directly protect against atherosclerosis by removing cholesterol from artery wall macrophages (see Tall, A. R., et al., J. Clin. Invest. 110:899-904, 2002; Oram, J. F., et al., Arterioscler. Thromb. Vasc. Biol. 23:720-727, 2003). Other studies have reported that HDL protects against LDL oxidative modification, which is believed to be central to the initiation and progression of atherosclerosis (see, e.g., Parthasarathy, S., et al., Biochim. Biophys. Acta 1044:275-283, 1990; Barter, P. J., et al., Circ Res 95:764-772, 2004). However, while HDL/LDL ratios have been correlated with risk for cardiovascular disease on an overall population, HDL and/or LDL measurements have not been reliable indicators of risk at an individual level.
Animal studies indicate that one important mechanism by which HDL protects against development of atherosclerosis involves reverse cholesterol transport in which HDL accepts cholesterol from macrophage foam cells in the artery wall and transports it back to the liver for excretion. HDL's cardioprotective effects may also depend on its anti-inflammatory properties. Indeed, HDL contains multiple acute phase response proteins, protease inhibitors and complement regulatory proteins (Vaisar, T., et al., J Clin Invest 117(3):746-756 (2007). Although HDL-cholesterol (HDL-C) levels are widely used to assess the risk for CAD, studies with genetically engineered animals convincingly demonstrate that changes in HDL metabolism can promote atherosclerosis by pathways that are independent of plasma levels of HDL-C. Also, the failure of recent clinical trials of a therapy that elevates HDL-C levels suggests that HDL can become dysfunctional in humans.
In accordance with the foregoing, in one aspect, a method of screening a mammalian test subject to determine if the subject is at risk to develop, or is suffering from, cardiovascular disease. The method comprises detecting a measurable feature of at least two biomarkers present in an HDL subfraction, or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the subject. The measurable features of the at least two biomarkers selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof, are then compared to a reference standard that is derived from measurements of the corresponding biomarkers present in comparable HDL subfractions or complexes isolated from biological samples obtained from a control population, such as a population of apparently healthy subjects. A difference in the measurable features of the at least two biomarkers between the test subject's sample and the reference standard, such as an average value from the control population, is indicative of the presence or risk of developing CAD in the subject. In some embodiments, the method further comprises determining whether the subject is exhibiting symptoms related to CAD.
The methods of this aspect of the invention are useful to screen any mammalian subject, including humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. A human subject may be apparently healthy or may be diagnosed as having a low HDL:LDL ratio and/or as being at risk for CAD based on certain known risk factors such as high blood pressure, high cholesterol, obesity, or genetic predisposition for CAD. The methods described herein are especially useful to identify subjects that are at high risk of developing CAD in order to determine what type of therapy is most suitable and to avoid potential side effects due to the use of medications in low risk subjects. For example, prophylactic therapy is useful for subjects at some risk for CAD, including a low fat diet and exercise. For those at higher risk, a number of drugs may be prescribed by physicians, such as lipid-lowering medications as well as medications to lower blood pressure in hypertensive patients. For subjects at high risk, more aggressive therapy may be indicated, such as administration of multiple medications.
In order to conduct sample analysis, a biological sample containing HDL is provided to be screened, including, but not limited to, whole blood or blood fractions (e.g., serum), bodily fluid, urine, cultured cells, tissue biopsies, or other tissue preparations. In some embodiments of the method of the invention, the biological samples include total HDL (density=about 1.06 to about 1.21 g/mL) or protein complexes that are isolated in this density range. In some embodiments of the method, a complex containing apoA-I and/or apoA-II is isolated from the biological sample. In other embodiments of the method of the invention, an HDL2 subfraction (density=about 1.06 to about 1.125 g/mL) is isolated from the biological sample prior to analysis. The HDL2 fraction may be isolated using any suitable method, such as, for example, through the use of ultracentrifugation, as described in Example 1.
In some embodiments, one or more of the biomarkers apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, including apoA-I oxidized at methionine residues and/or other HDL-associated peptides and/or proteins are isolated by liquid chromatography, affinity chromatography, or antibody-based methods from biological samples such as, but not limited to, blood, plasma, serum, urine, tissue, or atherosclerotic lesions.
As described in Examples 1-5, the present inventors have used matrix-assisted laser desorption mass spectrometry (MALDI-MS) to investigate the HDL proteome through the use of tryptic digestion. It was determined that the use of pattern recognition with two powerful linear algebraic techniques principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) could distinguish between tryptic digested HDL2 subfractions generated from control and CAD subjects at a high level of specificity and selectivity, as described in Example 2. Tandem mass spectrometry of informative mass features used to distinguish between normal and CAD subjects revealed a set of biomarkers for CAD as shown in TABLE 2 which include apoA-I (SEQ ID NO:1), apoA-II (SEQ ID NO:2), apoB-100 (SEQ ID NO:3), Lp(a) (SEQ ID NO:4), apoC-I (SEQ ID NO:5), apoC-III (SEQ ID NO:6), SAA4 (SEQ ID NO:7) and ApoE (SEQ ID NO:8), and peptide fragments and measurable features thereof.
The informative features that were identified that are useful to distinguish between normal and CAD subjects fall into the following classes: (1) increased levels of particular peptides/proteins in CAD subjects as compared to normal controls, for example, peptides derived from Lp(a) and/or apoC-III as shown in TABLE 3 and TABLE 4; (2) decreased levels of particular peptides/proteins in CAD subjects as compared to normal controls, for example, peptides derived from apoC-I as shown in TABLE 3 and TABLE 5; (3) post-translational modifications of particular peptides/proteins in CAD subjects as compared to normal controls, for example oxidation of M112 in apoA-I as shown in TABLE 3 and TABLE 6; and (4) altered conformational structure of particular peptides/proteins in CAD subjects as compared to normal controls, for example, apoA-I as shown in FIG. 7 and described in Example 5.
These results demonstrate that HDL isolated from subjects with CAD is selectively enriched in oxidized amino acids and certain proteins, and that the distinct cargo carried by the lipoprotein in subjects with clinically significant CAD may be assessed in a mammalian subject to determine his or her risk for developing CAD, the presence of CAD, and/or the efficacy of treatment of the subject for CAD. Therefore, the identification of peptides/proteins that are present in HDL of subjects suffering from CAD in amounts or structures that differ from normal subjects provide new biomarkers which are useful in assays that are prognostic and/or diagnostic for the presence of CAD and related disorders. The biomarkers may also be used in various assays to assess the effects of exogenous compounds for the treatment of CAD.
In one embodiment of this aspect of the invention, at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises an increased amount of at least one of the biomarkers in the HDL subfraction of the biological sample selected from the group consisting of apoA-I, apoB-100, apoC-III, and Lp(a), or portions and/or derivatives thereof, in comparison to the reference standard. For example, as demonstrated in Example 3, TABLE 3, and TABLE 4, tryptic peptides have been identified from apoA-I, apoB-100, apoC-III, and Lp(a) that were increased in HDL2 of CAD subjects as compared to normal control subjects. As shown in Examples 1 and 2, these peptides with increased frequency in CAD subjects are informative features for the prognosis and/or diagnosis of CAD.
In another embodiment of this aspect of the invention, at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises a decreased amount of at least one of the biomarkers in the HDL subfraction of the biological sample selected from the group consisting of apoA-I and apoC-I, or portions and/or derivatives thereof, in comparison to the reference standard. For example, as demonstrated in Example 3, TABLE 3, and TABLE 5, tryptic peptides have been identified from apoA-I and apoC-I that were decreased in HDL2 of CAD subjects as compared to normal control subjects. As shown in Examples 1 and 2, these peptides with decreased frequency in CAD subjects are informative features for the prognosis and/or diagnosis of CAD.
In another embodiment of the invention, at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises a post-translational modification of a peptide derived from apoA-I in the HDL subfraction of the biological sample, in comparison to the reference standard. For example, as demonstrated in Example 4 and TABLE 6, it has been determined that the oxidation state of apoA-I at M112 is indicative of the presence of CAD.
In the practice of the methods of the methods of this aspect of the invention, a measurable feature of at least two biomarkers (such as at least 3, at least 4, at least 5, or at least 6) selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III is detected. In accordance with this aspect of the invention, proteins having at least 90% identity (such as at least 95% identical, or at least 98% identical) with apoA-I (SEQ ID NO:1), apoA-II (SEQ ID NO:2), apoB-100 (SEQ ID NO:3), Lp(a) (SEQ ID NO:4), apoC-I (SEQ ID NO:5), and apoC-III (SEQ ID NO:6), and peptides derived therefrom, may be used as biomarkers for CAD, which are present at a differential level in CAD subjects as compared to normal control subjects. Peptide fragments derived from SEQ ID NOS: 1, 2, 3, 4, 5, and 6 may also be used as biomarkers, such as peptides from about 4 amino acids to at least about 20 amino acids or more. Representative peptide fragments that may be used as biomarkers in which an increased amount of the biomarker in HDL2 is indicative of the presence or risk of CAD include the peptides with positive regression vector values shown in TABLE 3 and TABLE 4. Representative peptide fragments that may be used as biomarkers in which a decreased amount of the biomarker in HDL2 is indicative of the presence or risk of CAD include the peptides with negative regression vector values shown in TABLE 3 and TABLE 5.
The presence and/or amount of the two or more HDL-associated biomarkers in a biological sample comprising total HDL, or a subfraction thereof, may be determined using any suitable assay capable of detecting the amount of the one or more biomarkers. Such assay methods include, but are not limited to, mass spectrometry, liquid chromatography, thin layer chromatography, fluorometry, radioisotope detection, affinity detection, and antibody detection. Other detection paradigms may optionally be used, such as optical methods, electrochemical methods, atomic force microscopy, and radio frequency methods (e.g., multipolar resonance spectroscopy). Optical methods include, for example, microscopy, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, and transmittance.
In one embodiment, the presence and amount of one or more HDL-associated biomarkers is determined by mass spectrometry. In accordance with this embodiment, biological samples may be obtained and used directly, or may be separated into total HDL or an HDL2 subfraction. The HDL-associated proteins are digested into peptides with any suitable enzyme such as trypsin, which cleaves adjacent to lysine (K) or arginine (R) residues in proteins. The peptides are then analyzed by a mass spectrometry method such as MALDI-TOF-MS or M/MS (solid phase), liquid chromatography (LC)-MS or MS/MS, μLC-ESI-MS/MS, and iTRAQTM ICAT, or other forms of isotope tagging. Any suitable method may be used for differential isotope labeling of proteins and/or peptide, such as the use of a compound or isotope-labeled compound that reacts with an amino acid functional group. Label-specific fragment ions allow one to quantify the differences in relative abundance between samples. For example, one useful approach to achieve quantitative results is the use of MALDI TOF/TOF or QTOF mass spectrometers and iTRAQTM, a commercially available stable isotope labeling system (Applied Biosystems, Foster City, Calif.). The iTRAQTM labeling system allows selective labeling of up to four different samples which are distinguished from one another in the mixture by MS/MS analysis.
By way of representative example, the method of MALDI-TOF-MS/MS involves the following steps. The samples are prepared and separated with fluidic devices, such as microfluidic devices, and spotted on a MALDI plate for laser-desorption ionization. Mass spectra are taken every few seconds, followed by isolation of the most intense peptide ions, or the peptide ions of interest (e.g., one derived from specific peptides), fragmentation by collisions with an inert gas, and recording of a mass spectrum of the fragments. This fragment mass spectrum, known as MS/MS spectrum, tandem mass spectrum, or MS2 spectrum, consists mainly of N- and C-terminal fragments of the peptide ions at the amide bonds, called b ions and y ions, respectively. The spectra are then matched to sequence databases, as further described in Example 3.
In a typical application of MS analysis, proteins in a biological sample are reduced, alkylated, digested into peptides with trypsin, and analyzed using multidimensional liquid chromatography and tandem mass spectrometry (MS/MS). Tryptic peptides are then subjected to multidimensional chromatography in concert with MS/MS analysis. In multidimensional chromatography, the first chromatographic dimension typically involves separation of digested peptides on a strong cation exchange column. The peptides are then typically separated through a reverse-phase column with increasing concentrations of acetonitrile and then introduced into the source of the mass spectrometer or fractionated directly onto a MALDI sample plate. Tandem mass spectra may be acquired in the data-dependent mode on an ion-trap, QTOF or MALDI-TOF/TOF instrument. The most abundant peaks from a survey scan are submitted to tandem MS analysis. In other applications, peaks that differ in intensity between samples of interest (e.g., a control population of apparently healthy subjects and subjects with established CVD) are selected from the MS or MS/MS spectra by a suitable method such as pattern recognition, cluster analysis, or relative abundance (see Rocke, D. M, Semin Cell Dev Biol 15:703-13, 2004; Ghazalpour, A., et al., Lipid Res 45:1793-805, 2004). The collection of tandem mass spectra may be submitted for a database search against a database (e.g., the Human International Protein Index (IPI) database, using the SEQUEST search engine (see Kersey, P. J., et al., “The International Protein Index: An Integrated Database for Proteomics Experiments,” Proteomics 4:1985-1988, 2004)), using software programs such as PeptideProphet, (Nesvizhskii, A. I., et al., Anal. Chem. 75:4646-4658, 2003) and ProteinProphet (Yan, W., et al., Mol. Cell. Proteomics 3:1039-1041, 2004) in order to refine peptide and protein identification.
To achieve semiquantitative results, protein abundance is estimated by the number of MS/MS spectra, the number of peptides detected, or by the percent of the protein sequence covered in the analysis. Quantitative results can be obtained with ICAT isotope tagging, iTRAQTM isotope labeling, or other modifications or peptides involving stable isotopes. Label-specific ions or fragment ions allow quantification of differences between samples based on their relative abundance.
Mass spectrometry detection methods may include the use of isotope-labeled peptides or proteins. In accordance with one example of this detection method, as described by Zou, H., et al., Cell 107:715-726, 2001, a tryptic peptide is chosen from a protein of interest. The tryptic peptide is then synthesized to incorporate one or more stable isotope-labeled amino acids. The native peptide and the synthetic-labeled peptide share physical properties including size, charge, hydrophobicity, ionic character, and amenability to ionization. When mixed, they elute together chromatographically, migrate together electrophoretically, and ionize with the same intensity. However, they differ in molecular weight from as little as 1 to over 10 Daltons, depending on which stable isotope amino acid is chosen for incorporation. The native peptide and the synthetic peptide are easily distinguishable by mass spectrometry. The synthetic peptide is used in an assay by adding a known amount of the synthetic peptide to a biological sample. In another example of this detection method, an isotope-labeled protein is prepared by a suitable method, such as by using a bacterial expression system and growing the bacteria on medium enriched with 15N-Nitrate or other isotope-labeled nutrients. The isotope-labeled peptide or protein is added to the sample containing native proteins and the mixture is then digested and analyzed by mass spectrometry as described herein. Extracted ion chromatograms or selected ion chromatograms or peak ratios in a full scan mass spectrum are then generated for the native peptide and the synthetic peptide. The quantity of the native peptide is then calculated using ratios of ion current or peak ratios.
Another detection method that utilizes labeled peptide fragments is isotope-coded affinity tagging (ICAT). This technique, as described in Gygi, S. P., et al., Nature Biotech. 17:994-999, 1999, involves the use of isotope tags that covalently bind to specific amino acids (cysteines) within a protein of interest. For example, the tag may contain three functional elements including a biotin tag (used during affinity capture), an isotopically encoded linker chain (such as an ether linkage with either eight hydrogens or eight deuteriums), and the reactive group, which binds to and modifies the cysteine residues of the protein. The isotope tag is used in an assay by labeling a control sample with the light version of the tag and labeling a test sample with the heavy version of the tag. The two samples are then combined, enzymatically digested, and the labeled cysteinyl residues may be captured using avidin affinity chromatography. The captured peptides are then analyzed by mass spectrometry, which can determine the relative abundance for each peptide-pair.
In another embodiment, antibodies are used in an immunoassay to detect one or more biomarkers in accordance with the method of this aspect of the invention. Such immunoassays may comprise an antibody to one or more of the biomarkers. The antibody is mixed with a sample suspected of containing the biomarker and monitored for biomarker-antibody binding. For example, the biomarker can be detected in an enzyme-linked immunosorbent assay (ELISA), in which a biomarker antibody is bound to a solid phase, such as a chip, and an enzyme-antibody conjugate is used to detect and/or quantify the biomarker(s) present in a sample.
In another aspect, the present invention provides a method of screening a mammalian subject to determine if the subject is at risk to develop, or is suffering from, or is recovering from a cardiovascular disease, the method comprising detecting an alteration in the conformational structure of apoA-I present in the HDL subfraction of a biological sample obtained from the test subject in comparison to a reference standard, wherein a difference in the conformation of the apoA-I between the biological sample from the subject and the reference standard is indicative of the presence or risk of cardiovascular disease in the subject.
In order to conduct sample analysis, a biological sample containing HDL is provided to be screened. Any HDL containing sample may be utilized with the methods described herein, including but not limited to whole blood or blood fractions (e.g., serum), bodily fluid, urine, cultured cells, biopsies or other tissue preparations. In some embodiments, the biological samples include total HDL (density=about 1.06 to about 1.21 g/mL) or protein complexes that are isolated in this density range. In some embodiments, an HDL2 subfraction (density=about 1.06 to about 1.125 g/mL) is isolated from the biological sample prior to analysis. In some embodiments, the HDL subfraction may be isolated by affinity isolation with polyclonal antibodies against apoA-I, the major protein in HDL or with polyclonal antibodies raised against other HDL associated proteins.
As described in Example 5, and shown in FIG. 7, it was determined that two tryptic peptides originating from N-terminal regions of apoA-I were significantly increased in the HDL subfraction of CAD subjects as compared to normal controls, while one tryptic peptide originating from the C-terminal region of apoA-I was significantly decreased. Although these N-terminal and C-terminal peptides are distant in the apoA-I sequence, when mapped to the double-belt model of the lipid-associated HDL particle apoA-I (Davidson, W. S, et al., J. Biol. Chem. 282(31):22249-22253, 2007, or the spherical HDL particle apoA-I model, the peptides displaying significant changes in CAD subjects were found to be in close proximity, as discussed in Example 5.
The conformation of apoA-I may be determined using any suitable method, such as by digesting the HDL subfraction of the biological sample with trypsin, followed by mass spectrometry analysis to measure the presence and/or amount of the tryptic fragments of apoA-I as compared to a reference standard, such as apoA-I isolated from normal control subjects. For example, the reference standard could be an exogenous isotopically labeled apoA-I which serves as an internal reference to which the intensity of individual peptides derived from apoA-I from the HDL subfraction of the biological sample would be related by a first ratio (i.e., apoA-I peptide from biological test sample/apoA-I peptide from reference standard). This first ratio would then be compared to a second ratio (i.e., apoA-I peptide from healthy control sample/apoA-I peptide from reference standard) to detect a difference in the amount of apoA-I peptides in the tested sample relative to the expected ratio in a healthy control sample, thereby indicating an altered apoA-I conformation.
In another example, the conformation of apoA-I may be determined by circular dichroism (CD), or with a monoclonal antibody that specifically detects the altered conformation of apoA-I. Methods of generating an antibody specific to an altered conformation of apoA-I are well known in the art, for example see Marcel, Y. L., et al., “Lipid Peroxidation Changes the Expression of Specific Epitopes of Apolipoprotein A-I.,” J. Biol. Chem. 264(33):19942-50, Nov. 25, 1989; Milthorp P, et al., “Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens,” Arteriosclerosis 6(3):285-96, May-June 1986; Marcel, Y. L., et al, “Monoclonal antibodies and the characterization of apolipoprotein structure and function,” Prog Lipid Res 23(4):169-95, 1984; and Weech, P. K., et al., “Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants,” Biochim Biophys Acta 835(2):390-401, Jul. 9, 1985., and Marcel Y. L. et al., “The epitopes of apolipoprotein A-I define distinct structural domains including a mobile middle region,” J. Biol. Chem. 266(6):3644-53, 1991.
In another aspect, the invention provides a method for diagnosing and/or assessing the risk of CAD in a subject, comprising determining changes in a biomarker profile comprising the relative abundance of at least one, two, three, four, five, ten or more biomarkers present in the HDL fraction of a biological sample from a test subject as compared to the predetermined abundance of the at least one, two, three, four, five, ten or more biomarkers from a reference population of apparently healthy subjects. The biomarkers are selected from biomarkers set forth in TABLE 3, TABLE 4, and TABLE 5. The biomarker profile may optionally include an internal reference standard that is expected to be equally abundant in subjects with CAD and apparently healthy subjects.
In another aspect, the present invention provides a method for determining the efficacy of a treatment regimen for treating and/or preventing CAD by monitoring a measurable feature of at least two biomarkers selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof in a subject during treatment for CAD. The treatment for CAD varies depending on the symptoms and disease progression. The general treatments include lifestyle changes, medications, and may include surgery. Lifestyle changes include, for example, weight loss, a low saturated fat, low cholesterol diet, reduction of sodium, regular exercise, and a prohibition on smoking. Medications useful to treat CAD include, for example, cholesterol-lowering medications, antiplatelet agents (e.g., aspirin, ticlopidine, clopidogrel), glycoprotein IIb-IIIa inhibitors (such as abciximab, eptifibatide or tirofiban), or antithrombin drugs (blood-thinners such as heparin) to reduce the risk of blood clots. Beta-blockers may be used to decrease the heart rate and lower oxygen use by the heart. Nitrates, such as nitroglycerin, are used to dilate the coronary arteries and improve blood supply to the heart. Calcium-channel blockers are used to relax the coronary arteries and systemic arteries and thus reduce the workload for the heart. Medications suitable for reducing blood pressure are also useful to treat CAD, including ACE inhibitors, diuretics, and other medications.
The treatment for cardiovascular disease may include surgical interventions such as coronary angioplasty, coronary atherectomy, ablative laser-assisted angioplasty, catheter-based thrombolysis, mechanical thrombectomy, coronary stenting, coronary radiation implant, coronary brachytherapy (delivery of beta or gamma radiation into the coronary arteries), and coronary artery bypass surgery.
In another aspect, the present invention provides assays and kits comprising one or more detection reagents for determining susceptibility or presence of cardiovascular disease in a mammalian subject based on the detection of at least one measurable feature of at least one biomarker in a biological sample, an HDL subfraction thereof, or a complex containing apoA-I or apoA-II isolated from the biological sample. The biomarker is detected by mixing a detection reagent that detects at least one biomarker associated with CAD with a sample containing HDL-associated proteins (either an HDL subfraction or a complex containing apoA-I or apoA-II) and monitoring the mixture for detection of the biomarker with a suitable detection method such as spectrometry, immunoassay, or other method. In one embodiment, the assays are provided as a kit. In some embodiments, the kit comprises detection reagents for detecting at least two, three, four, five, ten or more HDL-associated biomarkers in biological samples from a test subject.
The kit also includes written indicia, such as instructions or other printed material for characterizing the risk of CAD based upon the outcome of the assay. The written indicia may include reference information or a link to information regarding the predetermined abundance of the at least one, two, three, four, five, ten or more HDL-associated biomarkers from a reference population of apparently healthy subjects and an indication of a correlation between the abundance of one or more HDL-associated biomarkers and the risk level and/or diagnosis of CAD.
The detection reagents may be any reagent for use in an assay or analytical method, such as mass spectrometry, capable of detecting at least one measurable feature of at least one biomarker selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III. In another embodiment, the detection reagents include proteins with peptides identical to those of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, such as peptides provided in TABLE 3. A variety of protocols for measuring the relative abundance of the biomarkers may be used, including mass spectrometry, ELISAs, RIAs, and FACs, which are well known in the art.
In one embodiment, the detection reagent comprises one or more antibodies which specifically bind one or more of the biomarkers provided in TABLE 3, TABLE 4, or TABLE 5 that may be used for the diagnosis and/or prognosis of CAD characterized by the relative abundance of the biomarker in the serum, or an HDL subfraction thereof. Standard values for protein levels of the biomarkers are established by combining biological samples taken from healthy subjects. Deviation in the amount of the biomarker between control subjects and CAD subjects establishes the parameters for diagnosing and/or assessing risk levels, or monitoring disease progression. The biomarkers and fragments thereof can be used as antigens to generate antibodies specific for the CAD biomarkers for use in immunodiagnostic assays. Purified samples of the biomarkers comprising the amino acid sequences shown in TABLE 3, TABLE 4, and TABLE 5 may be recovered and used to generate antibodies using techniques known to one of skill in the art.
In another embodiment, the detection reagent comprises isotope-labeled peptides, such as one or more of the peptides described in TABLE 3, TABLE 4, and TABLE 5 that correspond to the biomarker to be detected. In accordance with this embodiment, the kit includes an enzyme, such as trypsin, and the amount of the biomarker in the tryptic digest of the sample is then quantified by isotope dilution mass spectrometry. The labeled peptides may be provided in association with a substrate, and the assay may be carried out in a multiplexed format. In one embodiment, a multiplexed format includes isotope-labeled peptides for at least two or more of the HDL-associated biomarkers described herein that are enriched in HDL of subjects with established CAD. The peptides are quantified of all the HDL-associated peptides in a biological sample obtained from a test subject using a technique such as isotope dilution mass spectrometry. The detection and quantification of multiple HDL-associated biomarker proteins may be used to increase the sensitivity and specificity of the assay to provide an accurate risk assessment and/or diagnosis of the presence of CAD in the test subject.
In one embodiment of the kit, the detection reagent is provided in association with or attached to a substrate. For example, a sample of blood, or HDL subfraction thereof, may be contacted with the substrate, having the detection reagent thereon, under conditions that allow binding between the biomarker and the detection reagent. The biomarker and/or the detection reagent are then detected with a suitable detection method. The substrate may be any suitable rigid or semirigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles, and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels, and pores to which the polypeptides are bound. For example, a chip, such as a biochip, may be a solid substrate having a generally planar surface to which a detection reagent is attached. For example, a variety of chips are available for the capture and detection of biomarkers, in accordance with the present invention, from commercial sources such as Ciphergen Biosystems (Fremont, Calif.), Packard BioScience Company (Meriden, Conn.), Zyomyx (Hayward, Calif.), and Phylos (Lexington, Mass.). An example of a method for producing such a biochip is described in U.S. Pat. No. 6,225,047. The biomarkers bound to the substrates may be detected in a gas phase ion spectrometer. The detector translates information regarding the detected ions into mass-to-charge ratios. Detection of a biomarker also provides signal intensity, thereby allowing the determination of quantity and mass of the biomarker.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.
Example 1
This example demonstrates that subjects may be successfully classified as normal control or coronary artery disease (CAD) subjects by analyzing the proteomic profile of HDL2 tryptic peptides using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) tandem mass spectrometry (MALDI-TOF-MS) and subjecting the results to principal component analysis (PCA), a well-established pattern recognition method.
Rationale:
The overall approach in this study was to isolate HDL2 from control and CAD subjects, analyze a tryptic digest of HDL proteins by MALDI-TOF-MS, and use pattern recognition of the full scan mass spectra to classify subjects as either CAD subjects or control subjects.
Methods:
Sample isolation and preparation: All protocols involving human subjects were approved by the Human Studies Committees at the University of Washington. Blood samples were collected from 20 apparently healthy adult males and from 18 male patients with established CAD after an overnight fast. Blood samples were anticoagulated with EDTA. All subjects were male and matched for age and HDL cholesterol (HDL-C) levels. The CAD subjects had documented vascular disease, with symptoms consistent with angina and abnormal Q waves on their EKG or at least one stenotic lesion (>50% occlusion on coronary angiography). These CAD subjects were clinically stable, at least three months had elapsed since their acute coronary syndrome, and they had not taken lipid-lowering drugs for the six weeks prior to blood collection. The control subjects were apparently healthy and had no known history of CAD, were not hyperlipidemic, had no family history of premature CAD, and were not receiving any lipid-lowering therapy. None of the control subjects smoked cigarettes, had liver or renal disease, were diabetic, or had received lipid-lowering medications for at least six weeks before blood was collected.
The clinical characteristics of the two subject populations are summarized below in Table 1.







TABLE 1







CLINICAL CHARACTERISTICS OF STUDY SUBJECTS


















Choles-
Triglyc-






Age

terol
erides
HDL-C
LDL-C


Number
Status
(yr)
% Male
(mg/dl)
(mg/dl)
(mg/dl) 
(mg/dl)





20
Control 
57
100
197 (13)
104 (29)
42 (8) 
134 (14)




(6)







18
CAD
57
100
223 (27)
146 (67)
41 (8) 
160 (25)




(6)









It is noted that although levels of plasma LDL and triglycerides were higher in the CAD subjects than in the control subjects, the two groups were otherwise well-matched for known risk factors for vascular disease.
HDL isolation: HDL2 (d=1.063 to 1.125 g/mL) was isolated from plasma obtained from the blood samples by sequential density ultracentrifugation, according to the methods described in Mendez, A. J., et. al., J. Biol. Chem. 266:10104-10111, 1991. Protein concentration of HDL was determined using the Bradford assay (BioRad, Hercules, Calif.) with albumin as the standard.
Tryptic Digestion: HDL2 was digested for 60 minutes with trypsin (1:50 w/w trypsin/HDL, sequencing grade trypsin, Promega WI) in 100 mM ammonium bicarbonate buffer in 80% aqueous acetonitrile (Strader, M. B., et al., Anal Chem 78(1):125-134, 2006). Digestion was terminated by addition of trifluoroacetic acid (TFA) to 1% final concentration.
The protein concentration of the HDL2 digest was adjusted to 100 ng/μL with matrix solvent (70% acetonitrile, 0.1% TFA), and 0.5 μl of the digest was deposited on a MALDI target plate. Dried spots were overlaid with 0.5 μL of MALDI matrix (5 mg/mL alpha-cyano-4-hydroxy-cinnamic acid (CHCA) in matrix solvent.
Mass spectrometric analysis. Mass spectra were acquired on a matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) tandem mass spectrometer (Applied Biosystems 4700 Proteomics Analyzer), operated in the reflection mode. Raw spectra were: (i) baseline-corrected and centroided using algorithms supplied by the manufacturer (ABI 4700 Explorer software, version 3.5); and (ii) internally mass calibrated using 5 tryptic fragments of apolipoprotein AI (apoA-I). The centroided spectra were then exported, using T2Extractor 5 (http://www.proteomecommons.org/archive/1114637208624/) for further analysis. It was determined that internal calibration afforded mass accuracy better than 5 ppm across the acquisition mass range.
For pattern recognition analysis, a single mass spectrum was generated from at least 80 sub-spectra generated randomly from different sites across the sample spot, each sampled with 25 laser shots, for a total of 2,000 shots. To exclude low intensity and saturated sub-spectra, only those with an ion current ranging from 30 to 80×104 cps were used to produce the mass spectrum.
The analytical precision of the different steps was evaluated by acquiring multiple spectra from: (1) the same MALDI spot; (2) multiple MALDI spots of the same tryptic digest; (3) multiple tryptic digests of the same sample; and (4) tryptic digestion of the same sample carried out on different days. As shown in FIGS. 9A-D, precision analysis of individual mass channels showed excellent reproducibility of the spectra from the same spot (FIG. 9A), multiple spots (FIG. 9B), parallel digestions (FIG. 9C), and interday digestion (FIG. 9D). The data indicated that precision was improved by averaging several spectra from the same spot. Thus, for the PLS-DA analysis, four spectra from the same spot were averaged to generate a master spectrum used for subsequent analyses.
Processing of MS Spectra: MATLAB (version 7.0, MathWorks Inc.) was used for pattern recognition analysis. The full scan mass spectrum of each sample was transformed into a vector format suitable for pattern recognition based on linear algebra by placing the signals in bins that ranged from m/z 800 to m/z 5,000. To ensure that every spectrum had the same mass channels, bin sizes were increased linearly over this range to yield 45,920 bins or channels per spectrum. After binning, data vectors were aligned to remove single bin shifts that occurred when signals were near the bin boarder. A threshold of 1/10,000 of the spectrum's total signal was used to remove baseline noise, and the spectra were aligned. For PLS-DA the data were separated into calibration and test sets prior to preprocessing to avoid overfitting. After alignment and filtering, 2,338 channels contained signals.
PCA Analysis: Processed MS spectra were then subjected to principal component analysis (PCA) and PLS-DA (Beebe, K. R., et al., Chemometrics: A practical guide. New York: Wiley—Interscience; 1998; and International Publication No. WO2006/083852 incorporated by reference herein in its entirety). PCA was used to assess the reproducibility of MALDI plate spotting and digestion and the sensitivity of MALDI-TOF-MS to changes in HDL protein composition. The latter was tested by mixing variable amounts of HDL2 isolated from CAD and control subjects, as shown in FIG. 8.
Validation of the Pattern Recognition Model: In order to test the ability of pattern recognition to distinguish between CAD and control HDL, PCA analysis was performed after mixing variable proportions of HDL2 isolated from control and CAD subjects. Mass spectra of 6 pairs of randomly chosen CAD and control samples were mixed in ratios of 1:0, 1:3, 1:1, 3:1, and 0:1. In separate experiments, blinded test samples from CAD or control subjects mixed at the same ratios were also included for the study.
Results:
PCA is a powerful linear algebraic technique for identifying factors that differentiate populations in a complex data set (Martens, H., et al., Multivariate Calibration. New York: John Wiley & Sons, 1989; Marengo, E., et al., Proteomics 5(3):654-666 (2005); Lee, K. R., et al., Proteomics 3(9):1680-1686 (2003); Natale, C. D., et al., Biosensors and Bioelectronics 18(10):1209-1218 (2003)). Importantly, this unsupervised data reduction method creates pattern recognition models without a priori assumptions regarding relationships between individual samples, (Beebe, K. R., et al., Chemometrics: A practical Guide. New York: Wiley-Interscience 1998).
PCA was initially used to test the ability of pattern recognition to distinguish between CAD and control HDL after mixing variable proportions of HDL2 isolated from control and CAD subjects. The results of this analysis are shown in FIG. 8, where the square symbols and triangle symbols represent different pairs of CAD and control samples mixed at different ratios. As shown in FIG. 8, the bottom left corner of the graph shows control samples and the upper right corner of the graph shows CAD samples. Circles around the symbols represent a group of spectra from different mixed ratios. As shown in FIG. 8, the control and CAD subjects were well separated by PCA analysis. When the relative proportion of HDL2 protein derived from control and CAD subjects in each sample was varied, there was a clear shift in the location of each sample on the PCA plot, as shown in FIG. 8, indicating that the method is sensitive to differences in the protein composition of HDL. Furthermore, the tight clustering of replicate spectra of the sample demonstrates the precision of this method.
These results demonstrate that subjects can be classified as CAD subjects based on the protein composition of their HDL2 which differs substantially as compared to the protein composition of HDL2 isolated from control subjects. These results further demonstrate that HDL2 from subjects may be successfully classified into CAD or control subjects based on the MALDI-TOF-MS and PCA-based pattern profiling described. Use of tryptic peptides significantly enhances the precision and probability of identifying proteins and post-translational modifications and allows rapid analysis. Furthermore, as shown in FIG. 9, the tight clustering of replicate spectra demonstrates the precision of the analytical method. Thus, PCA provides a fast, simple, exploratory, and qualitative measure of differences in the protein cargo of HDL2.
Example 2
This example demonstrates that subjects may be successfully classified into CAD or normal control subjects by analyzing tryptic digests of HDL2-associated proteins by MALDI-TOF-MS using a highly precise pattern recognition linear algebraic algorithm, partial least squares determination analysis (PLS-DA).
Rationale:
Although PCA is a powerful technique for detecting and visualizing differences in patterns, it does not provide quantitative measures for predicting the disease status of individual samples. Therefore, another powerful linear algebraic technique, partial least squares discriminate analysis (PLS-DA) was used to develop a quantitative approach to classifying subjects with regard to CAD disease status. PLS-DA was selected rather than other pattern recognition techniques (such as K-nearest neighbor and Support Vector Machine) because it is well suited to analyzing the quantitative information in a mass spectrum which contains multiple independent signals as well as signals with significant redundancy and signals with incomplete selectivity.
Methods:
Sample isolation and preparation. HDL2 fractions were isolated from the blood plasma of CAD and control subjects and digested with trypsin for 60 minutes, as described in Example 1. A sample from each subject was individually analyzed using MALDI-TOF MS as described in Example 1.
Processing of MS Spectra using Partial Least Squares Discriminate Analysis (PLS-DA) analysis. Matlab (version 7.0, Mathworks Inc.) was used for pattern recognition analysis. The full scan mass spectrum of each sample was transformed to a vector format suitable for pattern recognition based on linear algebra by placing the signals in bins that ranged from m/z 800 to m/z 5,000. To ensure that every spectrum had the same mass channels, bin sizes were increased linearly over this range to yield 45,920 bins or channels per spectrum. After binning, data vectors were aligned to remove single bin shifts that occurred when signals were near the bin boarder. A threshold of 1/10,000 of the spectrum's total signal was used to remove baseline noise, and the spectra were aligned. For PLS-DA, the data were separated into calibration and test sets prior to preprocessing to avoid overfitting. After alignment and filtering, 2,338 channels contained signals.
Preprocessed MS spectra were then subjected to PLS-DA. PLS-DA is a supervised pattern recognition technique. It uses two sets of data, such as training sets with defined groups (such as cases vs. controls) to “supervise” the creation of a pattern recognition model (Barker, M., et al., Journal of Chemometrics 16:166-173 (2003)), which is subsequently applied to a second test set of samples of unknown status. Thus, PLS-DA can be used to determine if a new proteomics sample belongs to previously defined sample classes. Furthermore, it can reveal relationships among sample classes and identify features distinguishing the classes, and ultimately the corresponding proteins. Most importantly, PLS-DA yields a single discriminant score that quantifies similarity of the tested spectrum with the model and can be used to predict the disease status of individual samples (CAD or control).
PLS-DA models were built with a dummy response matrix containing discrete numerical values (1 or −1) for each class as described in International Publication No. WO2006/083853. In the present analysis, “1” represented the CAD class and “−1” represented the control class. For each sample being classified, the PLS-DA model then produced a discriminant value, which was termed the “proteomics CAD risk score” or “ProtCAD” score. The ProtCAD risk scores thus generated were used to predict disease status of the remaining control and CAD subjects (validation group) as described in International Publication No. WO2006/083853, hereby incorporated by reference. To provide a quantitative estimate of the performance of the PLS-DA model, two approaches were used to provide a quantitative estimate of the performance: (1) Random permutation analysis; and (2) Leave-one-out analysis.
Random Permutation Analysis: When data from only a small number of subjects are used to build a complex pattern recognition model, predictions are often affected by the selection of the calibration subjects. Therefore, a permutation analysis was used to test the ProtCAD score's ability to predict disease status. In each step of this analysis, the subjects were assigned to two groups: a calibration group and a validation group, each composed of ten randomly selected control subjects and nine CAD subjects. The calibration group was used to build a PLS-DA model, which was then used to predict the ProtCAD score for each subject in the validation group. This process was repeated 7,777 times to determine the precision of the PLS-DA predictions, as described in International Publication No. WO2006/083853.
Receiver operating characteristics (ROC) curves: Nonparametric empirical receiver operating characteristic (ROC) curves were constructed from the ProtCAD risk score (Pepe, M. S., The Statistical Evaluation of Medical Tests for Classification and Prediction, New York, Oxford University Press, 2003). Sensitivity and specificity were calculated from the known class identity of each subject in the validation group. Area under the curve (AUC) calculations was determined using the trapezoidal rule (Fawcett, T., “An Introductory ROC Analysis,” Pattern Recognition Letters 27:861-874, 2006). For each permutation, one ROC curve was generated, and by plotting the sensitivity (fraction of positive results) against specificity (the fraction of negative results), ROC quantitatively assessed the accuracy of the predictive test. A quantitative PLS-DA model based on full scan mass spectra of HLD2 from a calibration group randomly selected subjects predicted CAD status in the validation group, with an average ROCAUC of 0.9. ROCAUC of 0.5 represents chance discrimination, whereas ROCAUC 1.0 represents perfect discrimination. For a CAD diagnostic test, an ROCAUC of 0.7-0.8 is generally considered acceptable, and values over 0.8 are considered excellent.
Leave-one-out ProtCAD prediction: In order to use the maximum number of available subjects, a leave-one-out approach was utilized to build a more powerful PLS-DA model, as described in International Publication No. WO2006/083853. The ProtCAD score for each subject was determined using a model built from the remaining subjects (e.g., 17 CAD and 19 controls). To predict the disease status of a subject, a ROC curve was first constructed and then the value of the ProtCAD score was compared to a threshold value corresponding to a selected sensitivity and selectivity on the ROC curve.
In preliminary experiments, it was determined that CAD predictions based on the entire mass spectrum outperformed predictions based on the ten most selective signals (as determined by PLS-DA, data not shown). Full spectrum PLS-DA can identify signals in regions normally associated with low selectivity and help identify outlier samples (e.g., problems with data acquisition from MS analysis, sample handling) or marked variations in sample protein composition (e.g., genetic variations in apoA-I, post-translational modifications). Such outliers would be overlooked by techniques that use only selected features of the spectrum. Therefore, all of the information in the full scan mass spectra was used to build models.
Results:
Two approaches were used to assess the ability of PLS-DA to distinguish the proteomic fingerprints of HDL isolated from control and CAD subjects. First, a PLS-DA model was built using data from randomly selected control and CAD subjects. Then the model was tested for its ability to predict disease status in a second set of subjects. The PLS-DA models were built with a dummy response matrix containing discrete numerical values for each class using ten control and nine CAD subjects (the calibration set) that were randomly selected from the 20 control and 18 CAD subjects. The PLS-DA model was then used to predict the disease status of the remaining ten control and nine CAD subjects (the validation set). For each sample in the validation set the approach produced a discriminant score, which was termed the “Proteomics CAD risk score” (ProtCAD risk score).
Random permutation analysis was used to provide a quantitative estimate of the performance of the PLS-DA model that was used to build the discriminant termed the Proteomics CAD risk score (ProtCAD risk score). The ProtCAD score was then used to predict the CAD status of the validation group (the remaining nine CAD and ten control subjects). A total of 7,777 random permutations were used to construct the ROC curve. FIG. 1 presents graphical results demonstrating the receiver operating characteristic (ROC) curve of the prediction of cardiovascular disease (CAD) status based on random permutation analysis. By plotting sensitivity (the fraction of true positive results, shown in the y-axis) against specificity (the fraction of true negative results, shown in the x-axis), the ROC curve quantitatively assesses the accuracy of a predictive test. As shown in FIG. 1, the average ROC curve shows an area-under-the-curve of 0.880±0.097 (mean, SD. N=7,777 iterations), indicating that PLS-DA analysis can predict disease status with high sensitivity and specificity.
FIG. 2 graphically illustrates the prediction of CAD status by the proteomics CAD risk score “ProtCAD risk score” using a partial least squares discriminate analysis (PLS-DA) model built using a calibration group. Using a sensitivity of 80%, the ProtCAD risk score of each subject in the validation group at each permutation was used to predict their CAD status. The results in FIG. 2 demonstrate that the ProtCAD score generated using the PLS-DA model is able to distinguish CAD and control subjects with high selectivity (p-value of 0.0001%). Using a clinically acceptable sensitivity of 80%, (see Pepe, M. S., The Statistical Evaluation of Medical Tests for Classification and Prediction, New York: Oxford University Press, 2003), the average PLS-DA model predicted CAD status with 76% specificity, as shown in FIG. 2.
The level of specificity shown in FIG. 2 corresponds to an odds ratio of 12.7, i.e., the odds ratio of the PLS-DA model for predicting CAD status here was 12.7 at 80% sensitivity and 76% specificity. These results demonstrate the power of this analytical approach for identifying subjects at risk for CAD.
In the second approach, disease status was predicted by using PLS-DA models built with the leave-one-out method as described in International Publication No. WO2006/083853. This strategy allowed the use of all available subjects for the analysis, which would be expected to yield the strongest predictive model. After systematically leaving out one subject at a time from the calibration set, the subject's ProtCAD score was predicted using a model built from the remaining samples.
FIG. 3 graphically illustrates the power of the ProtCAD risk score to discriminate between the CAD samples and healthy control samples based on leave-one-out analysis. The ProtCAD risk score was derived from PLS-DA analysis of MALDI-TOF-MS mass spectra of HDL tryptic digests, using a leave-one-out experiment for all 18 CAD and 20 control subjects. These ProtCAD scores distinguished the CAD and control subjects with high selectivity (p<0.0001), as shown in FIG. 3. Furthermore, the larger number of subjects in the calibration set improved diagnostic power. As shown in FIG. 10A, the ROC curve constructed for ProtCAD scores from the leave-one-out approach showed an area-under-the-curve of 0.94 and a maximum odds ratio of 68. From the leave-one-out ProtCAD risk score ROC curve, we determined a threshold corresponding to 90% sensitivity (ProtCAD threshold=−0.06). Using this threshold, the model correctly classified 16 of 18 CAD subjects and 19 of 20 control subjects.
Therefore, these results demonstrate that pattern recognition analysis, when applied to MALDI-TOF-MS spectra of tryptic digests of HDL2, clearly demonstrate differences in the HDL proteomic signature of apparently healthy subjects and CAD subjects. Moreover, a quantitative PLS-DA model based on full scan mass spectra of HDL2 from a calibration group of randomly selected subjects predicted CAD status in the validation group, with an average ROCAUC of 0.9 (ROCAUC 0.5 represents chance discrimination, whereas ROCAUC 1.0 represents perfect discrimination).
For a CAD diagnostic test, an ROCAUC of 0.7 to 0.8 is generally considered acceptable, and values over 0.8 are considered excellent (see Pepe, M. S., The Statistical Evaluation of Medical Tests for Classification and Prediction, New York, Oxford University Press, 2003). Furthermore, the odds ratio of the PLS-DA model for predicting CAD status was 12.7 at 80% sensitivity and 76% specificity. When the model was built with data from a larger number of subjects using the leave-one-out method, the ProtCAD risk score distinguished subjects with an even higher odds ratio of 68. These results compare favorably with other single lipoprotein-associated risk factors identified in previous studies (Yusuf, S., et al., Lancet, 364(9438):937-952, 2004; Walldius, G., and I. Jungner, J. Intern. Med. 259(5):493-519, 2006; Walldius, G., and I. Jungner, Curr. Opin. Cardiol. 22(4):359-367, 2007).
The standard method for predicting CAD, the Framingham risk score, combines seven demographic, biochemical and medical factors to predict CAD risk (Wilson, P. W., et al., Circulation 97(18):1837-1847 (1998)). The Framingham risk scores ROCAUC ranges from 0.6-0.8 for predicting CAD risk over a ten year period. Its strongest predictors are age and sex, which are not modifiable risk factors. Moreover, LDL-C and HDL-C contribute little to the risk score in some models (Yusuf, S., et al., Lancet 364(9438):937-952, 2004; Walldius, G., et al., Curr Opin Cardiol 22(4):359-367, 2007; Walldius, G., et al., J Intern Med 259(5):493-519, 2006).
On the other hand, this example indicates that the HDL2 isolated from CAD subjects with its characteristic proteome profile is faster and more accurate at predicting risk with a ROCAUC of 0.9.
Conclusion:
As demonstrated herein, the protein composition of the HDL2 in subjects with CAD as well as the protein composition of HDL2 isolated from control subjects are different. Differences in the protein profiles can be accurately and quantitatively measured using the two different approaches together with the PLS-DA algorithm. These observations also show that PLS-DA analysis can correctly and with high sensitivity predict the status of a subject as a CAD subject or a control subject.
The methods of proteomic fingerprinting of HDL by MALDI-TOF-MS offer a number of important advantages for building classification models. First, it has been demonstrated that HDL is causally linked to CAD pathogenesis. Second, the HDL proteome is much simpler than the plasma proteome (which has been estimated to contain>104 different proteins and peptides with relative concentrations ranging over 12 orders of magnitude), which greatly facilitates MS analysis. Third, the interrogation of tryptic digests significantly enhances the precision of the mass spectrometric measurements, and thereby increases the probability of identifying proteins and post-translational modifications. In contrast to the methods described herein, surface-enhanced laser desorption ionization (SELDI) MS analysis, which has been widely used for pattern recognition, typically samples intact proteins, which makes it difficult to identify the proteins responsible for informative signals in quantitative models. SELDI MS also has a limited mass range and low mass resolution, which bias detection of informative features toward degraded and low MW proteins. Finally, the high mass accuracy of MALDI-TOF-MS facilitates the subsequent identification of proteins and posttranslational modifications by tandem MS. MALDI-TOF-MS of tryptic digests also greatly improves the precision of signals, which is important for pattern recognition analysis.
Previous studies using shotgun proteomics to investigate the protein composition of HDL3 using liquid chromatography in concert with electrospray ionization (ESI) to introduce peptides into the mass spectrometer (Vaisar, T., et al., J. Clin. Invest. 117(3):746-756 (2007)). In contrast, in the present study the peptides were ionized with MALDI. It is well established that ESI and MALDI ionize different classes of peptides with different efficiencies. For example, hydrophobic peptides are much more readily introduced into the gas phase by MALDI.
Example 3
This example describes the identification of proteins differentially present in HDL2 subfractions isolated from normal control and CAD subjects by tryptic peptide analyses of HDL2 fractions by tandem mass spectrometric (tandem MS) following PLS-DA based pattern recognition profiling.
Methods:
Sample isolation and preparation: HDL2 fractions were isolated from normal control and CAD subjects as described earlier in Example 1. Tryptic digests of HDL2 fractions were subjected to liquid chromatographic separation with direct application of the sample effluent from the liquid chromatograph onto a MALDI sample plate and subjected to MALDI-TOF/TOF tandem mass spectrometric analysis (LC-MALDI-TOF/TOF). Subjects were confirmed as either CAD or normal subjects by pattern recognition proteomic profiling of HDL2 proteins using PLS-DA, as described in Example 2.
The PLS-DA models are characterized by regression vectors which indicate channels on the m/z axis of a mass spectrum that differentiate the two sample classes.
FIG. 4 graphically illustrates the PLS-DA regression vectors (y-axis) of the leave-one-out PLS-DA model that distinguish CAD and control subjects. The x-axis (m/z) represents mass channels of the MALDI-TOF mass spectrum. Positive and negative features on the regression vector indicate an increase and decrease of the signals from CAD samples (and therefore relative amount of peptide present) relative to control samples. Each mass channel in the regression vector that had significant differences between CAD and normal subjects was called an informative feature.
Channels in the regression vectors with positive values correspond to the peptides (and indirectly to the proteins) with increased relative abundance in CAD samples. Channels with negative values in the regression vector have decreased abundance in CAD samples. As shown in FIG. 4, a subset of 13 informative features were identified with the most significant increase or decrease in CAD subjects as compared to normal control subjects in a full scan mass spectrum that contributed to the ability to differentiate CAD subjects from normal subjects. The peptides associated with these informative features were identified by tandem MS using the MALDI-TOF/TOF analyzer capable of MS and MS/MS analysis interfaced with an off-line capillary liquid chromatograph and coupled with a MALDI plate spotter. As described in International Publication No. WO2006/083853, chromatographic information may be used to more strongly validate that the peptide identified is actually producing the observed peak in the regression vector.
Identification of significant features by Liquid-Chromatography (LC) matrix-assisted laser desorption ionization (MALDI): To identify features that were enriched or depleted in the mass spectra of HDL isolated from CAD subjects, CAD HDL tryptic digests were fractionated by liquid chromatography and the peptide digest was subjected to MS/MS analysis by MALDI-TOF/TOF. A tryptic digest of HDL was injected onto a trap column (NanoTrap C18, LC Packings), washed, and eluted onto an analytical capillary HPLC column (PepMap C18, LC Packings) using an Ultimate 300 HPLC (LC Packings Inc.). Separation was achieved by linear gradient 5-50% B over 40 minutes (A-5% aqueous can, 0.1% TFA, B-80% aqueous can, 0.1% TFA). The eluent from the column was mixed with matrix (CHCA, 5 mg/ml in 70% AcN) containing internal standard peptides and spotted on-line (Shimadzu Accuspot MALDI plate spotter) on a MALDI target plate. Targeted MS/MS analysis of selected peptide ions was based on informative mass features of HDL proteomics fingerprints that were identified in the PLS-DA analysis. Peptides were identified by MASCOT database search (v2.0, Matrix Science) against the human SwissProt protein database with the following parameters; trypsin cleavage with up to two missed cleavages, methionine oxidation variable modification, precursor tolerance 15 ppm, and fragment ion tolerance 0.2 Da. Peptide matches were only accepted if the MASCOT probability based Mowse score identified the peptide with a very high score indicating a match to the database with >99% confidence.
Results:
It was determined that the relative abundance of HDL2 apolipoproteins was altered in CAD subjects compared to the controls.
One group of informative features arose from proteins in the HDL2 fraction that were differentially abundant in CAD and control subjects. As shown in FIG. 4, informative features with positive regression vector values were observed at channels 1081, 1226, 1440, 1715*, 1904, 2203, and 2661 m/z in the CAD subjects relative to the control subjects, indicating that the peptides (and therefore proteins) represented by these regression vectors at these channels were more abundant in CAD subjects.
As further shown in FIG. 4, informative features with negative regression vector values were observed at channels 852*, 1012, 1302, 1380, 1612, and 2645 m/z in the CAD subjects relative to the control subjects, indicating that the peptides (and therefore proteins) represented by these regression vectors at these channels were reduced in CAD subjects as compared to normal control subjects.
TABLE 2 provides a set of informative biomarkers corresponding to features from FIG. 4 that were identified using the targeted LC-MALDI-TOF/TOF approach.







TABLE 2







BIOMARKERS IDENTIFIED AS PROGNOSTIC AND/OR


DIAGNOSTIC INDICATORS OF CAD












Refseq ID




Protein
Numbers
SEQ ID NO:







apoA-I
NP_000030.1
SEQ ID NO: 1



apoA-II
NP_001634
SEQ ID NO: 2



apoB-100
NP_000375
SEQ ID NO: 3



Lp(a)
NP_005568.1
SEQ ID NO: 4



apoC-I
NP_001636.1
SEQ ID NO: 5



apoC-III
NP_000031.1
SEQ ID NO: 6



SAA4 (serum amyloid
NP_006503
SEQ ID NO: 7



A4-confirm)





apoE
NP_000032
SEQ ID NO: 8










Targeted tandem MS analysis was carried out to identify the peptides corresponding to the informative features shown in FIG. 4. The results are shown in TABLE 3.







TABLE 3







PEPTIDES IDENTIFIED AS INFORMATIVE FOR CAD













Magnitude in







Regression



SEQ ID


m/z
Vector
Peptide
Protein
start-stop
NO















861.5088
−11.679
ITLPDFR
apoB-100
2706-2712 
 9


999.5271
−5.9951
SVGFHLPSR
apoB-100
1325-1333 
10


1012.6055
−39.9366
AKPALEDLR
apoA-I
231-239
11


1013.5781
−21.0822
AKPALEDLR-I
apoA-I
231-239
11


1014.5921
−5.15
AKPALEDLR-II
apoA-I
231-239
11


1031.5333
−5.700
LSPLGEEMR
apoA-I
165-172
12


1032.524
−3.785
LSPLGEEMR-I
apoA-I
165-173
12


1033.5571
5.470
LSPLGEEMR-II
apoA-I
165-173
14


1033.5571
5.470
LQAEAFQAR
apoE
270-278
13


1047.4997
−6.987
LSPLGEEMoxR
apoA-I
165-173
12


1048.5057
−3.295
LSPLGEEMoxR-I
apoA-I
165-173
12


1049.5128
−1.862
LSPLGEEMoxR-II
apoA-I
165-173
12


1081.6043
22.482
LAAYLMLMR
apoB-100
559-567
15


1141.6155
2.912
HINIDQFVR
apoB-100
2101-2109
16


1156.6456
−3.813
SKEQLTPLIK
apoA-II
68-77
17


1156.6456
−3.813
SPAFTDLHLR
apoB-100
3980-3989
18


1157.6638
−22.261
LEALKENGGAR
apoA-I
202-212
19


1157.6638
−22.261
SKEQLTPLIK-I
apoA-II
68-77
17


1158.6367
−15.025
LEALKENGGAR-I
apoA-I
202-212
19


1158.6367
−15.025
SKEQLTPLIK-II
apoA-II
68-77
17


1159.6567
−5.85
LEALKENGGAR-II
apoA-I
202-212
19


1159.6567
−5.85
SKEQLTPLIK-III
apoA-II
68-77
17


1160.6312
−0.846
LEALKENGGAR-III
apoA-I
202-212
19


1160.6312
−0.846
SKEQLTPLIK-IV
apoA-II
68-77
17


1166.5888
−8.57
FRETLEDTR
apoB-100
2512-2520
20


1167.5691
−5.47
FRETLEDTR-I
apoB-100
2512-2520
20


1168.597
−1.685
FRETLEDTR-II
apoB-100
2512-2520
20


1169.579
−10.437
SLDEHYHIR
apoB-100
2211-2219
21


1170.6086
−7.161
SLDEHYHIR
apoB-100
2211-2219
21


1171.5924
−1.733
SLDEHYHIR
apoB-100
2211-2219
21


1178.6429
−6.301
VLVDHFGYTK
apoB-100
733-742
22


1179.6334
−5.424
VLVDHFGYTK-I
apoB-100
733-742
22


1199.6662
−6.224
VKSPELQAEAK
apoA-II
52-62
23


1200.6743
−4.688
VKSPELQAEAK-I
apoA-II
52-62
23


1201.6352
−4.73
VKSPELQAEAK-II
apoA-II
52-62
23


1201.6352
−4.73
LTISEQNIQR
apoB-100
335-344
24


1202.645
−2.364
VKSPELQAEAK-III
apoA-II
52-62
23


1202.645
−2.364
LTISEQNIQR-I
apoB-100
335-344
24


1226.547
31.827
DEPPQSPWDR
apoA-I
25-34
25


1227.5777
22.165
DEPPQSPWDR-I
apoA-I
25-34
25


1283.6171
17.33
WQEEMELYR
apoA-I
132-140
26


1284.6444
13.139
WQEEMELYR-I
apoA-I
132-140
26


1285.6211
4.945
WQEEMELYR-II
apoA-I
132-140
26


1286.5985
−1.058
WQEEMELYR-III
apoA-I
132-140
26


1299.5808
4.112
WQEEMoxELYR
apoA-I
132-140
26


1300.5688
4.532
WQEEMoxELYR-I
apoA-I
132-140
26


1301.6617
−16.664
THLAPYSDELR
apoA-I
185-195
27


1302.6514
−82.138
THLAPYSDELR-I
apoA-I
185-195
27


1302.6514
−82.138
LSPLGEEMRDR
apoA-I
165-175
28


1303.6417
−50.00
THLAPYSDELR-II
apoA-I
185-195
27


1303.6417
−50.00
LSPLGEEMRDR-I
apoA-I
165-175
28


1304.685
−25.542
KGNVATEISTER
apoB-100
196-207
29


1305.6769
−7.513
KGNVATEISTER-I
apoB-100
196-207
29


1306.6696
−0.949
KGNVATEISTER-II
apoB-100
196-207
29


1318.6407
2.632
LSPLGEEMoxRDR
apoA-I
165-175
28


1319.6432
1.355
LSPLGEEMoxRDR-I
apoA-I
165-175
28


1380.7137
−20.692
VQPYLDDFQKK
apoA-I
121-131
30


1381.7081
−15.84
VQPYLDDFQKK
apoA-I
121-131
30


1400.6834
6.728
DYVSQFEGSALGK
apoA-I
52-64
31


1401.6922
4.3536
DYVSQFEGSALGK-I
apoA-I
52-64
31


1402.7018
−2.083
DYVSQFEGSALGK-II
apoA-I
52-64
31


1403.7121
−4.059
DYVSQFEGSALGK-III
apoA-I
52-64
31


1404.7231
−2.216
DYVSQFEGSALGK-IIII
apoA-I
52-64
31


1410.748
−6.002
FQFPGKPGIYTR
apoB-100
4202-4213 
32


1411.7077
−12.606
KWQEEMELYR
apoA-I
131-140
33


1412.7244
−1.039
KWQEEMELYR-I
apoA-I
131-140
33


1412.7244
−1.039
DPDRFRPDGLPK
SAA4
117-128
34


1413.6854
−1.114
KWQEEMELYR-II
apoA-I
131-14-
33


1413.6854
−1.114
DPDRFRPDGLPK-I
SAA4
117-128
34


1414.7036
0.290
KWQEEMELYR-III
apoA-I
131-140
33


1414.7036
0.290
DPDRFRPDGLPK-II
SAA4
117-128
34


1415.7225
0.7011
KWQEEMELYR-IV
apoA-I
131-140
33


1415.7225
0.7011
DPDRFRPDGLPK-III
SAA4
117-128
34


1427.6644
11.458
KWQEEMoxELYR
apoA-I
131-140
33


1428.6927
9.069
KWQEEMoxELYR-I
apoA-I
131-140
33


1429.6645
5.938
KWQEEMoxELYR-II
apoA-I
131-140
33


1440.6864
48.538
NPDAVAAPYCYTR
Lp(a)
79-91
35


1441.6664
35.366
NPDAVAAPYCYTR-I
Lp(a)
79-91
35


1442.7047
16.693
NPDAVAAPYCYTR-II
Lp(a)
79-91
35


1488.7235
−10.607
MREWFSETFQK
apoC-I
64-74
36


1489.7361
−8.862
MREWFSETFQK-I
apoC-I
64-74
36


1490.6898
−3.125
MREWFSETFQK-II
apoC-I
64-74
36


1504.7079
−12.493
MoxREWFSETFQK
apoC-I
64-74
36


1505.7314
−12.066
MoxREWFSETFQK-I
apoC-I
64-74
36


1506.6954
−5.215
MoxREWFSETFQK-II
apoC-I
64-74
36


1568.8737
9.352
LAARLEALKENGGAR
apoA-I
198-212
37


1569.8781
6.904
LAARLEALKENGGAR-I
apoA-I
198-212
37


1585.8456
4.483
THLAPYSDELRQR
apoA-I
185-197
38


1586.8608
0.9348
THLAPYSDELRQR-I
apoA-I
185-197
38


1612.7768
−17.535
LLDNWDSVTSTFSK
apoA-I
70-83
39


1716.8928
6.669
DALSSVQESQVAQQAR
apoC-III
45-60
40


1717.8545
3.025
DALSSVQESQVAQQAR-I
apoC-III
45-60
40


1718.8855
−0.99
DALSSVQESQVAQQAR-II
apoC-III
45-60
40


1723.9809
2.583
QKVEPLRAELQEGAR
apoA-I
141-155
41


1723.9809
2.583
IVQILPWEQNEQVK
apoB-100
577-590
42


1724.9466
1.7167
QKVEPLRAELQEGAR-I
apoA-I
141-155
41


1724.9466
1.7167
IVQILPWEQNEQVK-I
apoB-100
577-590
42


1725.9818
1.707
QKVEPLRAELQEGAR-II
apoA-I
141-155
41


1725.9818
1.707
IVQILPWEQNEQVK-II
apoB-100
577-590
42


1775.9145
15.536
NLQNNAEWVYQGAIR
apoB-100
4107-4121
43


1776.9092
12.946
NLQNNAEWVYQGAIR-I
apoB-100
4107-4121
43


1777.9046
11.265
NLQNNAEWVYQGAIR-II
apoB-100
4107-4121
43


1904.9087
34.681
TPEYYPNAGLIMNYCR
Lp(a)
177-192
44


1905.8995
35.544
TPEYYPNAGLIMNYCR-I
Lp(a)
177-192
44


1906.8908
24.997
SEAEDASLLSFMQGYMK
apoC-III
21-37
45


1906.8908
24.997
TPEYYPNAGLIMNYCR-II
Lp(a)
177-192
44


1907.8826
12.840
SEAEDASLLSFMQGYMK-I
apoC-III
21-37
45


1907.8826
12.840
TPEYYPNAGLIMNYCR-III
Lp(a)
177-192
44


1922.8989
7.340
SEAEDASLLSFMoxQGYMK
apoC-III
21-37
45


2202.1435
41.389
LREQLGPVTQEFWDNLEK
apoA-I
 84-101
46


2203.2007
49.396
LREQLGPVTQEFWDNLEK-I
apoA-I
 84-101
46


2204.1703
35.316
LREQLGPVTQEFWDNLEK-II
apoA-I
 84-101
46


2205.1404
16.202
LREQLGPVTQEFWDNLEK-III
apoA-I
 84-101
46


2206.1991
6.574
LREQLGPVTQEFWDNLEK-IIII
apoA-I
 84-101
46


2645.4139
−7.042
VQPYLDDFQKKWQEEMELYR
apoA-I
121-140
47


2646.3664
−12.328
VQPYLDDFQKKWQEEMELYR-I
apoA-I
121-140
47


2647.4251
−9.890
VQPYLDDFQKKWQEEMELYR-II
apoA-I
121-140
47


2648.3783
−5.448
VQPYLDDFQKKWQEEMELYR-III
apoA-I
121-140
47


2661.3337
8.767
VQPYLDDFQKKWQEEMoxELYR
apoA-I
121-140
47


2662.3985
12.259
VQPYLDDFQKKWQEEMoxELYR-I
apoA-I
121-140
47


2663.3571
9.880
VQPYLDDFQKKWQEEMoxELYR-II
apoA-I
121-140
47


2664.4226
5.8323
VQPYLDDFQKKWQEEMoxELYR-III
apoA-I
121-140
47









The m/z values are peaks that were obtained for the markers using mass spectrometry system using the methods described herein.
As shown in TABLE 3, a marker may be represented at multiple m/z points in a spectrum. This can be due to the fact that multiple isotopes (represented in TABLE 3 as “I, II, III, IIII”) were observed, and/or that multiple charge states of the marker were observed, or that multiple isoforms of the marker were observed, for example, a post-translational modification such as oxidation. These multiple representations of a particular marker can be analyzed individually or grouped together. An example of how multiple representations of a marker may be grouped is that the intensities for the multiple peaks can be summed.
As shown below in TABLE 4 and TABLE 5, targeted tandem MS analysis identified the peptides corresponding to ten of the 13 informative features shown in FIG. 4 (i.e., most significant features that contributed to the PLS-DA model).







TABLE 4







INFORMATIVE FEATURES REPRESENTING INCREASED PROTEIN/PEPTIDE LEVELS IN CAD


SUBJECTS AS COMPARED TO NORMAL SUBJECTS













Magnitude
Protein






in
corresponding 


SEQ 


Channel
Regression
to identified
Protein

ID


m/z
Vector
peptides
Residues
Peptide Sequence
NO















1081.6043
+22.482
apo-B100
559-567
LAAYLMLMR
15


1226.547
+31.83
apo-Al
25-34
DEPPQSPWDR
25


1227.5777
+22.165
apo-AI
25-34
DEPPQSPWDR-I
25


1440.6864
+48.538
Lp(a)
79-91
NPDAVAAPYCYTR
35


1441.6664
+35.366
Lp(a)
79-91
NPDAVAAPYCYTR-I
35


1442.7047
+16.693
Lp(a)
79-91
NPDAVAAPYCYTR-II
35


1904.9087
+34.681
Lp(a)
177-192
TPEYYPNAGLIMNYCR
44


1905.8995
+35.544
Lp(a)
177-192
TPEYYPNAGLIMNYCR-I
44


1906.8908
+24.997
Lp(a)
177-192
TPEYYPNAGLIMNYCR-II
44


1907.8826
+12.840
Lp(a)
177-192
TPEYYPNAGLIMNYCR-III
44


1906.8908
+24.997
apoC-III
21-37
SEAEDASLLSFMQGYMK
45


1907.8826
+12.840
apoC-III
21-37
SEAEDASLLSFMQGYMK-I
45


1922.8989
+7.340
apoC-III
21-37
SEAEDASLLSFMoxQGYMK
45


2202.1435
+41.39
apoA-I
 84-101
LREQLGPVTQEFWDNLEK
46


2203.2007
+49.39
apoA-I
 84-101
LREQLGPVTQEFWDNLEK-I
46


2204.1703
+35.32
apoA-I
 84-101
LREQLGPVTQEFWDNLEK-II
46


2205.1404
+16.202
apoA-I
 84-101
LREQLGPVTQEFWDNLEK-III
46


2206.1991
+6.574
apoA-I
 84-101
LREQLGPVTQEFWDNLEK-IIII
46


2661.3337
+8.767
apoA-I
121-140
VQPYLDDFQKKWQEEM(Ox)ELY
47




(Met112ox)

R



2662.3985
+12.259
apoA-I
121-140
VQPYLDDFQKKWQEEM(Ox)ELY
47




(Met 112ox)

R-I



2663.3571
+9.880
apoA-I
121-140
VQPYLDDFQKKWQEEM(Ox)ELY
47




(Met112ox)

R-II



2664.4226
+5.8323
apoA-I
121-140
VQPYLDDFQKKWQEEM(Ox)ELY
47




(met112ox)

R-III









As shown above in TABLE 4, identification of the tryptic peptides associated with the positive regression vector values shown in FIG. 4 revealed that, surprisingly, two peptides identified at m/z 1440 to 1442 (SEQ ID NO: 35) and m/z 1904 to 1906 (SEQ ID 44) derived from apolipoprotein(a) (Lp(a)) were increased in HDL2 of CAD subjects, as compared to normal subjects. FIG. 5A graphically illustrates the strong positive informative feature in the PLS-DA regression vector at m/z 1440. As shown in FIG. 5C, the positive informative feature at m/z 1440 was identified by LC-MALDI-TOF/TOF MS/MS as corresponding to the peptide NPDAVAAPYCYTR (SEQ ID NO:35) which corresponds to amino acids 79-91 of Lp(a) (SEQ ID NO:4), with a MASCOT ion score of 86.46 (CI=100%). As shown in FIG. 5B, another strong positive informative feature in the PLS-DA regression vector at m/z 1904 was identified as corresponding to the peptide TPEYYPNAGLIMNYCR (SEQ ID NO:44), which corresponds to amino acids 177-192 of Lp(a) (SEQ ID NO:4).
As further shown in TABLE 4, tryptic peptides identified at m/z 1906-1922 (SEQ ID NO: 45) derived from apoC-III (SEQ ID NO:6) were increased in HDL2 of CAD subjects, as compared to normal subjects.







TABLE 5







INFORMATIVE FEATURES REPRESENTING DECREASED PROTEIN/PEPTIDE LEVELS IN CAD


SUBJECTS AS COMPARED TO NORMAL SUBJECTS













Magnitude
Protein






in
corresponding 


SEQ 


Channel
Regression
to Identified
Protein

ID


m/z
Vector
Peptides
Residues
Peptide sequence
NO:















1012.6055
−39.93
apoA-I
231-239
AKPALEDLR
11


1013.5781
−21.082
apoA-I
231-239
AKPALEDLR-I
11


1014.5921
−5.15
apoA-I
231-239
AKPALEDLR-II
11


1157.6638
−22.261
apoA-I
202-212
LEALKENGGAR
19


1158.6367
−15.025
apoA-I
202-212
LEALKENGGAR-I
19


1159.6567
−5.85
apoA-I
202-212
LEALKENGGAR-II
19


1160.6312
−0.846
apoA-I
202-212
LEALKENGGAR-III
19


1301.6617
−16.664
apoA-I
185-195
THLAPYSDELR
27


1302.6514
−82.138
apoA-I
185-195
THLAPYSDELR-I
27


1303.6417
−50.00
apoA-I
185-195
THLAPYSDELR-II
27


1302.6514
−82.138
apoA-I
165-175
LSPLGEEMRDR
28


1303.6417
−50.00
apoA-I
165-175
LSPLGEEMRDR-I
28


1380.7137
−20.69
apoA-I
121-131
VQPYLDDFQKK
30


1381.7081
−15.84
apoA-I
121-131
VQPYLDDFQKK-I
30


1488.7235
−10.607
apoC-I
64-74
MREWFSETFQK
36


1489.7361
−8.862
apoC-I
64-74
MREWFSETFQK-I
36


1490.6898
−3.125
apoC-I
64-74
MREWFSETFQK-II
36


1504.7079
−12.493
apoC-I
64-74
MoxREWFSETFQK
36


1505.7314
−12.066
apoC-I
64-74
MoxREWFSETFQK-I
36


1506.6954
−5.215
apoC-I
64-74
MoxREWFSETFQK-II
36


1612.7768
−17.53
apoA-I
70-83
LLDNWDSVTSTFSK
39


2645.4139
−7.042
apoA-I
121-140
VQPYLDDFQKKWQEEMELYR
47


2646.3664
−12.328
apoA-I
121-140
VQPYLDDFQKKWQEEMELYR-I
47


2647.4251
−9.89
apoA-I
121-140
VQPYLDDFQKKWQEEMELYR-II
47


2648.3783
−5.448
apoA-I
121-140
VQPYLDDFQKKWQEEMELYR-III
47









As shown above in TABLE 5, identification of the tryptic peptides associated with the negative regression vector values shown in FIG. 4 revealed several peptides from apoA-I and a peptide from apoC-I were decreased in HDL2 of CAD subjects compared to that of control subjects. The peptides derived from apoA-I (SEQ ID NO:1) that were identified as decreased in CAD subjects included SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:39 and SEQ ID NO:47, as shown in TABLE 5.
The negative regression vector at m/z 1504-1506 was identified as corresponding to the peptide MREWFSETFQK (SEQ ID NO: 36) which corresponds to amino acids 64-74 of ApoC-I (SEQ ID NO: 5).
Taken together, these results demonstrate that pattern recognition profiling performed on HDL2 isolated from CAD and control subjects show altered patterns of apoproteins present in the HDL2 fractions which fall into two classes: (1) increased levels of peptides/proteins in CAD subjects as compared to normal controls; or (2) decreased levels of peptides/proteins in CAD subjects as compared to normal controls.
The observation that levels of Lp(a) were found to be increased in CAD subjects in comparison to normal controls was a surprising result because Lp(a) has been shown to be associated with small dense low density lipoproteins (LDLs), and its association with HDLs in general, and HDL2 in particular, has not been previously shown. Thus, these results demonstrate that co-isolation of Lp(a) with HDL2 subfraction permits pattern recognition analysis of the HDL2 subfraction in the prediction, diagnosis, and prognosis of CAD subjects.
It was also observed that levels of apoC-III peptides were found to be elevated in CAD subjects, whereas those of apoC-I were decreased. In this regard, although not wishing to be bound by theory, it is noted that apoC-III inhibits lipoprotein lipase and the hepatic uptake of triglyceride-rich lipoproteins, which might promote an increase in atherogenic triglyceride-rich lipoproteins (see Ooi, E. M., et al., Clin Sci (Lond) 114:611-624, 2008. It is further noted that ApoC-I inhibits cholesterol ester transfer protein (CETP) (see Shachter, N. S., et al., Curr Opin Lipidol 12:297-304 (2001); Sparks, D. L., et al., J. Lipid Res 30:1491-1498, 1989. Thus, it is believed that alterations in apoC-I and apoC-III levels are likely contribute to lipid remodeling and the formation of pro-atherogenic HDL particles.
Therefore it is demonstrated that simultaneous profiling of these biomarkers in subjects using pattern recognition analysis may be used to aid in the diagnosis and prognosis of cardiovascular diseases in mammalian subjects.
Example 4
This example demonstrates that subjects may be successfully classified as CAD or control subjects based on the oxidation status of their HDL2 using PLS-DA based pattern recognition proteomic profiling.
Methods:
Sample preparation and analysis: HDL2 fractions were isolated from subjects, and samples from each individual subject were subjected to MALDI-TOF/TOF MS and PLS-DA analyses, as described in Example 2. Subjects were classified as either CAD or normal control subjects by pattern recognition proteomic profiling of HDL2 proteins using PLS-DA. The PLS-DA models were characterized by regression vectors as described in Example 3. The PLS-DA model regression vector analysis is centered on post-translationally modified peptides derived from apoA-I, the major protein in HDL2.
Results:
In addition to the first two groups of informative features (increased or decreased peptide levels in CAD subjects as compared to normal subjects) as described in Example 2, a third group of informative features in the PLS-DA model was identified that centered on post-translationally modified peptides derived from apoA-I (SEQ ID NO:1), the major protein in HDL. MS/MS analysis confirmed the presence of these peptide sequences in the HDL2 fraction and demonstrated that the methionine 112 residue had been converted to methionine sulfoxide (Met(O)).
As shown in FIGS. 6A-D and summarized below in TABLE 6, this third group of informative features included both native peptides KWQEEMELYR (SEQ ID NO:33) and VQPYLDDFQKKWQEEMELYR (SEQ ID NO: 47) and the corresponding oxidized peptides that contained methionine 112 (Met112). FIG. 6A graphically illustrates the negative regression vector at m/z 1411.7077 and the positive regression vector at m/z 1427.6644 which were identified as corresponding to the native form of the apoA-I peptide KWQEEMELYR (SEQ ID NO: 33), and the Met112 oxidized form KWQEEM(O)ELYR of SEQ ID NO:33, respectively, as shown in FIG. 6C (MASCOT ion score of 84.8, CI=100%).
FIG. 6B graphically illustrates the negative regression vector at m/z 2646.3664 and the positive regression vector at m/z 2662.3985 which were identified as corresponding to the native form of the apoA-I peptide VQPYLDDFQKKWQEEMELYR (SEQ ID NO:47), and the Met112 oxidized form VQPYLDDFQKKWQEEM(O)ELYR of SEQ ID NO:47, respectively, as shown in FIG. 6D (MASCOT ion score of 42.6, CI=99.96%).







TABLE 6







INFORMATIVE FEATURES REPRESENTING POSTTRANSLATIONALLY MODIFIED PEPTIDES


IN CAD SUBJECTS AS COMPARED TO NORMAL SUBJECTS













Magnitude in
Protein/





Channel
Regression
peptide


SEQ


m/z
Vector
location
Modification
Peptide Sequence
ID





1411.7077
−12.606
apoA-I
native
KWQEEMELYR
33




(131-140)





1427.6644
+11.458
apoA-I
oxidized M112
KWQEEM(O)ELYR
33




(131-140)
(MetOx)




2646.3664
−12.328
apoA-I
native
VQPYLDDFQKKWQEEMELYR-I
47




(121-140)





2662.3985
+12.259
apoA-I
oxidized M112
VQPYLDDFQKKWQEEM(O)ELYR-I
47




(121-140)
(MetOx)









Strikingly, as shown in FIG. 6 and summarized above in TABLE 6, the signals for the Met 112 oxidized (Met112(O)) apoA-I peptides (SEQ ID NO:33 and SEQ ID NO:47) were found to be increased in CAD subjects as compared to normal control subjects, while the levels of the corresponding native Met112 peptides (SEQ ID NO:33 and SEQ ID NO:47) were found to be decreased in CAD subjects as compared to normal control subjects.
It is noted however, that no difference in relative levels of other methionine containing native and oxidized peptides, such as those derived from apoC-I, were observed between normal controls and CAD subjects was observed in this analysis (data not shown), suggesting that the difference in levels of oxygenated Met112 did not result from ex vivo oxidation.
While not wishing to be bound by theory, oxidation has been proposed as one mechanism for generating dysfunctional HDL resulting in decreased reverse cholesterol transport, thereby disrupting normal cholesterol homeostasis. Lipid hydroperoxides and reactive intermediates produced by Myeloperoxidase (MPO) oxidize apoA-I. It has been shown that oxidation of methionine residues impairs apoA-I's ability to promote cholesterol efflux by the ABCA1 pathway (Shao, B., et al., J Biol Chem 281(14):9001-9004 (2006) and to activate LCAT, two key steps in removing cholesterol from lipid-laden macrophages. apoA-I co-localizes with HOCl oxidation adducts in human atherosclerotic tissues. MPO-produced HOCl is known to modify HDL in vivo. Antibodies specific for apoA-I and HOCl-modified proteins immunostained coronary arteries obtained from patients undergoing cardiac transplantation (O'Brien et al., Circulation 98:519-527, 1998). apoA-I co-localized with epitopes recognized by HOP-I antibody, which is specific for proteins oxidized by HOCl (Hazell et al., J. Clin. Invest. 97:1535-1544, 1996) in the intima of atherosclerotic lesions. The co-localization of HOCl-modified proteins with apoA-I suggests that HOCl oxidizes specific proteins in the human artery wall.
Oxidized HDLs are also present in the circulation of CVD patients, (International Publication No. WO2006/014628). Circulating HDL from cardiovascular patients has 8-times higher 3-chlorotyrosine than normal subjects. Levels of chlorinated HDL are elevated in the blood of humans suffering from clinically significant atherosclerosis. In addition, MPO-produced H2O2 is also capable of oxidizing methionines of apoA-I associated with HDL3 (International Publication No. WO2006/014628). These HDL3 subfractions are selectively enriched with oxidized amino acids.
Collectively, these observations support the conclusion that HDL2 from control and CAD subjects differ in their protein cargoes and levels of oxidized methionine residues. Because pattern recognition analysis makes no assumptions about the origins of the differential signals seen in the regression vectors for each sample, it provides a powerful tool for identifying post-translationally modified peptides that would be very difficult to identify using classic proteomic approaches. The results demonstrated in this example indicate that oxidized methionines (Met(O)) in apoA-I are detectable by pattern recognition profiling of an HDL2 subfraction. Since oxidation of methionine residues impairs apoA-I's ability to promote cholesterol efflux by the ABCA1 pathway (Shao, B., et al., “Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I,” J. Biol. Chem. 281:9001-4, 2006) and to activate Lecithin:Cholesterol Acyltransferase (LCAT) (Shao, B., et al., “Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I,” Proc. Natl. Acad. Sci. 2008, In press), oxidized apoA-I likely acts as a mediator of CAD, and serves as a useful biomarker for CAD. Thus, a subject may be evaluated for the presence of oxidized apoA-I (SEQ ID NO:1) to determine the risk, diagnosis, prognosis of CAD in the subject and/or to measure the efficacy of treatment of a subject suffering from CAD.
Example 5
This example demonstrates that the conformational structure of apoA-I in HDL2 subfractions is altered in CAD subjects as compared to the conformation structure of apoA-I in HDL2 subfractions of normal control subjects.
Rationale:
The structural conformation of apoA-I has been suggested to influence its ability to transfer cholesterol ester from HDL2 particles to scavenger receptor BI as part of reverse cholesterol transport and cholesterol ester clearance in the liver (de Beer, M. C., et al., J. of Lipid Res 42:309-313, February 2001). Contact between the N-terminal fold and the C-terminal domain of apoA-I has been suggested to stabilize the lipid-bound conformation of the protein. Since methionines in apoA-I are oxidized in CAD subjects, as demonstrated in Example 4, an experiment was carried out to determine if such post-translational modifications lead to local changes in the structural conformation of apoA-I. Alterations in a protein's local structure is said to affect susceptibility of the protein to proteolytic digestion, which in turn can affect the apparent abundance of peptides assessed by MS.
Methods:
Sample preparation and analysis: HDL2 fractions were isolated from subjects and treated with trypsin as disclosed in Example 1. The samples from each individual subject were subjected to MALDI-TOF/TOF MS, as described in Example 2. Subjects were classified as either CAD or normal subjects by pattern recognition proteomic profiling of HDL2 proteins using PLS-DA. The PLS-DA models were characterized by regression vectors as described in Example 3. The PLS-DA model regression vector analysis is centered on apoA-I peptides of HDL2. The differential signals reflecting the relative abundance of the trypsinized peptides was measured.
Results:
In addition to the three groups of informative features (increased or decreased peptide levels in CAD subjects as compared to normal subjects) as described in Example 2, and post-translationally modified peptides derived from apoA-I as described in Example 3, a fourth group of informative features in the PLS-DA model was identified based on the altered structural conformation of apoA-I present in the HDL2 subfraction of CAD subjects in comparison to the structural conformation of apoA-I present in the HDL2 subjection of normal subjects.
Informative features corresponding to tryptic peptides derived from the N-terminal and C-terminal regions of apoA-I (SEQ ID NO:1) were identified. FIG. 7 graphically illustrates the regression vector values (y-axis) for the amino acid sequence of apoA-I (x-axis).
It was determined that two tryptic apoA-I peptides originating from N-terminal regions of the mature protein (residues 1-10: DEPPQSPWDR (SEQ ID NO:48) and residues 60-77, LREQLGPVTQEFWDNLEK (SEQ ID NO:49) were significantly increased in CAD subjects as compared to normal controls, while one C-terminal region peptide (residues 207-215, AKPALEDLR (SEQ ID NO:50) was significantly decreased as compared to normal controls, as shown in FIG. 7. Also, a tryptic peptide (peptide 46-59: LLDNWDSVTSTFSK (SEQ ID NO:52) was apparently decreased in abundance. These observations suggest that tryptic digests of apoA-I in HDL isolated from control and CAD subjects give different patterns of peptides, perhaps because of conformational differences of the apoA-I in the two different classes of subjects. Indeed, although the above-referenced N-terminal peptides (SEQ ID NO:48 and SEQ ID NO:49) and C-terminal peptides (SEQ ID NO:49) are distant in apoA-I sequence, when mapped to the double-belt model of the lipid-associated apoA-I (Davidson, W. S., et al., J. Biol. Chem. 282(31):22249-22253 (2007)) or spherical HDL particle apoA-I model (Davidson W. S. PNAS 2008, in press), the peptides displaying significant changes in CAD subjects were found to be in close proximity (data not shown).
Additionally, it was determined that the peptides (residues 97-107, VQPYLDDFQKK SEQ ID NO: 51) proximal to Met112 was significantly decreased in the CAD samples (FIG. 7), as is the peptide containing Met112, SEQ ID NO:33.
It was recently proposed that contact between the globular N-terminal fold and the C-terminal fold of apoA-I stabilizes the lipid-bound conformation of the protein. It is important to note that alterations in a proteins' local structure can effect susceptibility to proteolytic digestion, which in turn can affect the apparent abundance of peptides in a MS analysis. As demonstrated in this example, the differential levels of N-terminal and C-terminal apoA-I peptides indicates that the secondary and/or tertiary conformations at the N and C-termini of apoA-I differ in the HDL2 of CAD subjects as compared to normal control subjects. Further in this regard, as described in Example 4 and summarized in TABLE 6, it was also determined that levels of apoA-I peptides containing Met(O)112 were elevated in the HDL2 of CAD subjects concomitantly with a decrease in Met112 peptides in the CAD subjects. The peptides directly adjacent to the peptides containing Met112 also displayed significant changes in CAD subjects as compared to normal controls. While not wishing to be bound by theory, these observations suggest that oxidation of methionine residues in apoA-I is increased in CAD subjects and such oxidation may lead to local changes in the conformation of the apoA-I protein which can be detected by tryptic digestion followed by analysis by mass spectrometry.
The results described in this example demonstrate that the altered conformation of apoA-I at its N- and C-termini is detectable using PLS-DA-based pattern recognition profiling. Changes in relative abundance of certain tryptic peptides demonstrate that apoA-I exists in altered secondary and/or tertiary conformation in HDL2 subfractions of CAD subjects compared to control subjects. Thus, dysfunctionality of HDL2 of CAD subjects likely results from changes in the proteome profile and conformation of the associated HDL2 proteins. These results demonstrate that pattern recognition profiling using tryptic peptides of HDL2 subfractions from subjects can be used to determine the conformation status of apoA-I in order to classify subjects as normal or CAD patients.
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

## Claims

1. A method of screening a mammalian subject to determine if the subject is at risk to develop, or is suffering from, cardiovascular disease, the method comprising detecting a measurable feature of at least two biomarkers in an HDL subfraction, or in a complex containing apoA-I or apoA-II isolated from a biological sample obtained from the subject, wherein the at least two biomarkers are selected from the group consisting of apoA-I, apoA-II, apoB-100, Lp(a), apoC-I, and apoC-III, combinations or portions and/or derivatives thereof, and comparing the measurable features of the at least two biomarkers from the biological sample to a reference standard, wherein a difference in the measurable features of the at least two biomarkers from the biological sample and the reference standard is indicative of the presence or risk of cardiovascular disease in the subject.

2. The method of claim 1, wherein the HDL subfraction of the biological sample is the HDL2 subfraction.

3. The method of claim 1, wherein the biological sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a tissue sample, a bodily fluid sample, and a urine sample.

4. The method of claim 1, wherein at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises an increased amount of at least one of the biomarkers in the HDL subfraction or in the complex containing apoA-I or apoA-II isolated from the biological sample selected from the group consisting of apoA-I, apoB-100, apoC-III and Lp(a), or portions and/or derivatives thereof, in comparison to the reference standard.

5. The method of claim 4, wherein the biomarker is apoA-I, or a portion or derivative thereof.

6. The method of claim 4, wherein the biomarker is apoC-III or a portion or derivative thereof.

7. The method of claim 4, wherein the biomarker is Lp(a) or a portion or derivative thereof.

8. The method of claim 1, wherein at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises a decreased amount of at least one of the biomarkers in the HDL subfraction or in the complex containing apoA-I or apoA-II isolated from the biological sample selected from the group consisting of apoA-I and apoC-I, or portions and/or derivatives thereof, in comparison to the reference standard.

9. The method of claim 8, wherein the biomarker is apoA-I, or a portion or derivative thereof.

10. The method of claim 8, wherein the biomarker is apoC-I, or a portion or derivative thereof.

11. The method of claim 1, wherein at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises a post-translational modification of a peptide derived from apoA-I in the HDL subfraction or complex isolated from the biological sample, in comparison to the reference standard.

12. The method of claim 11, wherein the post-translational modification of apoA-I is oxidation of at least one Methionine residue.

13. The method of claim 1, wherein at least one of the measurable features indicative of the presence or risk of cardiovascular disease comprises an altered structural conformation of apoA-I in the HDL subfraction of the biological sample, in comparison to a reference standard.

14. The method of claim 1, wherein the measurable features of the at least two biomarkers from the biological sample are determined using mass spectrometry analysis.

15. The method of claim 14, wherein the mass spectrometry analysis is performed on a tryptic digestion of the HDL subfraction or complex isolated from the biological sample.

16. The method of claim 14, wherein the mass spectrometry analysis is carried out with a matrix-assisted laser desorption ionization (MALDI) mass spectrometer or LCMS.

17. The method of claim 1, wherein the cardiovascular disease is the predisposition to myocardial infarction, atherosclerosis, coronary artery disease, peripheral artery disease, heart failure, or stroke.

18. The method of claim 1, wherein the measurable features of the at least two biomarkers in the HDL subfraction or complex isolated from the biological sample are detected using at least one antibody specific to each of the at least one of the two biomarkers.