Compositions and methods for purification and detection of HDL and APOA1

Abstract

The present invention provides methods, kits and compositions for purifying HDL molecules from a sample (e.g., a blood sample) using HDL labeling molecules comprising an HDL lipophilic core binding peptide (e.g., a portion of ApoA1) and an affinity tag. The present invention also provides methods, kits and compositions for detecting non-fragmented ApoA1 by mass spectrometry. The present invention further provides methods, kits and compositions for labeling HDL molecules in a sample with a detectably labeled ApoA1 molecule so that the ratio of the detectably labeled ApoA1 molecule to the native ApoA1 protein can be determined.

Description

[0001] This application is a divisional application of Chinese patent application 201580030344.X (PCT application number PCT / US2015 / 030949), whose application date is May 15, 2015 and whose invention name is “Compositions and methods for purification and detection of HDL and APOA1”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 61 / 993,696, filed May 15, 2014, which is incorporated herein by reference. Field of the Invention

[0004] The present invention provides methods, kits, and compositions for purifying HDL molecules from a sample (e.g., a blood sample) using HDL labeling molecules comprising an HDL lipophilic core-binding peptide (e.g., a portion of ApoA1) and an affinity tag. The present invention also provides methods, kits, and compositions for detecting non-fragmented ApoA1 using mass spectrometry. The present invention further provides methods, kits, and compositions for labeling HDL molecules in a sample with detectably labeled ApoA1 molecules, such that the ratio of detectably labeled ApoA1 molecules to native ApoA1 protein can be determined. background

[0005] Serum lipoproteins comprise a heterogeneous population of lipid-protein complexes that can be grouped into broad classes based on differences in particle density related to lipid and protein content: very low (VLDL), low (LDL), and high (HDL) density. VLDL and LDL are primarily composed of lipids, while high-density lipoproteins have a higher protein content (approximately 50%). The density of LDL ranges from 1.006 to 1.063 g / ml, while the density of HDL and HDL-like particles ranges from 1.063 to 1.21 g / ml. The classical method for separating HDL from VLDL and LDL employs continuous density ultracentrifugation using potassium bromide salt solutions prepared to have densities within the range of each lipoprotein class. One disadvantage of these methods for preparing purified HDL is that they require at least two extended ultracentrifugation steps. The first step to separate VLDL and LDL from HDL requires an 18-hour ultracentrifugation spin in a KBr salt solution with d = 1.063 g / ml. The buoyant VLDL and LDL are concentrated in the upper layer of the salt gradient and can be easily removed, leaving the less buoyant HDL concentrated in the lower layer along with other heavier proteins. HDL is then separated from other lipid-free serum proteins by performing a second ultracentrifugation step in a d = 1.21 g / ml KBr salt solution for 21 hours. The HDL is buoyant in this density salt solution, so at the end of the centrifugation, the upper layer of the gradient contains mainly HDL, leaving other plasma proteins in the bottom portion. This continuous density gradient ultracentrifugation procedure is the "standard of excellence" for the isolation of HDL. However, the extended time required for the two ultracentrifugation steps and the need for multiple density adjustments clearly limit the throughput of the procedure. SUMMARY OF THE INVENTION

[0006] The present invention provides methods, kits, and compositions for purifying HDL molecules from a sample (e.g., a blood sample) using an HDL labeling molecule comprising an HDL lipophilic core-binding peptide (e.g., a portion of ApoA1) and an affinity tag. In certain embodiments, the HDL purification is rapid (e.g., less than 1 hour) and allows determination of at least one cardiovascular risk factor (e.g., cholesterol level, oxidation state of ApoA1, etc.). The present invention also provides methods, kits, and compositions for detecting non-fragmented ApoA1. The present invention further provides methods, kits, and compositions for labeling HDL molecules in a sample with a detectably labeled ApoA1 molecule so that the ratio of the detectably labeled ApoA1 molecule to the native ApoA1 protein can be determined.

[0007] In some embodiments, provided herein are methods for producing a purified sample, the methods comprising: a) mixing an initial sample (e.g., a sample that is or is not ApoB / LDL-depleted) containing a group of HDL molecules (e.g., mature HDL molecules) and non-HDL biomolecules with a group of HDL marker molecules to produce a mixed sample, wherein each of the HDL molecules comprises: i) an HDL lipophilic core and ii) a plurality of HDL lipoproteins, and wherein each of the HDL marker molecules comprises: i) an HDL lipophilic core-binding peptide, and ii) an affinity tag; b) incubating the mixed sample such that at least some of the HDL marker molecules bind to at least some of the HDL molecules, thereby producing a group of labeled HDL molecules; and c) purifying at least a portion of the group of labeled HDL molecules away from the non-HDL biomolecules (and unlabeled HDL molecules) to produce a purified sample, wherein the purification comprises contacting the mixed sample with a group of capture molecules specific for the affinity tag.

[0008] In certain embodiments, the HDL marker molecule is added to the initial sample such that the ratio of labeled ApoA1 molecules to unlabeled ApoA1 molecules is about 1:10-10:1, 1:5-4:1, or about 1:3-3:1, or about 1:2-2:1; or about 1: 1. In certain embodiments, the initial sample is a serum sample, and the amount of HDL marker molecule added to the serum sample is about 0.1 mg-4 mg / ml serum sample, or about 0.5 mg-2 mg / ml serum sample, or about 1 mg / ml serum sample.

[0009] In some embodiments, provided herein are compositions comprising: a) a population of HDL marker molecules comprising: i) at least a portion of ApoA1 or an ApoA1 mimetic that is capable of binding HDL, and ii) an affinity tag; and b) a population of unlabeled, wild-type ApoA1 molecules; wherein the ratio of the HDL marker molecules to the unlabeled molecules present in the composition is from 1:2 to 2:1.

[0010] In certain embodiments, the composition further comprises human serum, whole blood, plasma, or a reconstituted HDL sample. In further embodiments, the human serum is non-LDL-depleted human serum, whole blood, or plasma. In other embodiments, the affinity tag does not contain unpaired electrons. In additional embodiments, the unlabeled, wild-type ApoAl molecule is a portion of an HDL molecule.

[0011] In some embodiments, provided herein are compositions comprising: a) a non-LDL-deficient blood, plasma, or serum sample; and b) a group of HDL marker molecules, each comprising: i) an HDL lipophilic core-binding peptide, and ii) an affinity tag. In certain embodiments, the HDL lipophilic core-binding peptide comprises an HDL binding region of apolipoprotein AI (ApoA1), and wherein the non-LDL-deficient blood, plasma, or serum sample comprises unlabeled ApoA1 molecules. In additional embodiments, the HDL marker molecules are present in the non-LDL-deficient blood, plasma, or serum sample such that the ratio of the HDL marker molecules to the unlabeled ApoA1 molecules in the composition is 1:2-2:1.

[0012] In some embodiments, provided herein are compositions comprising an HDL marker molecule comprising: a) an HDL lipophilic core-binding peptide, and b) an affinity tag, wherein the affinity tag does not contain unpaired electrons.

[0013] In certain embodiments, provided herein are compositions comprising labeled HDL molecules, wherein the labeled HDL molecules comprise: a) an HDL molecule comprising: i) an HDL lipophilic core and ii) a plurality of HDL lipoproteins, and b) an HDL marker molecule comprising: i) an HDL lipophilic core-binding peptide and ii) an affinity tag, wherein the affinity tag does not contain unpaired electrons, and wherein the HDL lipophilic core-binding peptide binds to the HDL lipophilic core.

[0014] In a further embodiment, provided herein are compositions comprising: a) an HDL marker molecule comprising: i) an HDL lipophilic core binding peptide, and ii) an affinity tag; and b) a population of capture molecules, wherein the capture molecules are specific for the affinity tag.

[0015] In certain embodiments, provided herein are kits and systems comprising: a) an HDL marker molecule comprising: i) an HDL lipophilic core-binding peptide, and ii) an affinity tag; and b) a population of capture molecules, wherein the capture molecules are specific for the affinity tag. In certain embodiments, the HDL marker molecule is in a first container, and wherein the population of capture molecules is in a second container.

[0016] In certain embodiments, the HDL lipophilic core binding peptide comprises the HDL binding region of apolipoprotein AI (ApoA1). In certain embodiments, the lipophilic core binding peptide comprises a portion of human ApoA1, such as amino acid residues 188-243 of human ApoA1. In other embodiments, the plurality of HDL lipoproteins in each of the HDL molecules comprises a first and a second native ApoA1 protein, and wherein when the labeled HDL molecule binds to the HDL molecule, at least one of the HDL marker molecules displaces (or binds to the lipophilic core together with the first and second native ApoA1 molecules) the first native ApoA1 protein in each of the HDL molecules. In further embodiments, the HDL lipophilic core binding peptide comprises at least a portion of ApoA1 or an ApoA1 mimetic.

[0017] In further embodiments, the HDL lipophilic core-binding peptide comprises the HDL binding region of apolipoprotein A-II (ApoA2) (e.g., human ApoA1). In additional embodiments, the HDL lipophilic core-binding peptide comprises at least a portion of ApoA2 or an ApoA2 mimetic. In certain embodiments, the HDL lipophilic core-binding peptide comprises the HDL binding region of apolipoprotein E (ApoE) (e.g., human ApoE). In additional embodiments, the HDL lipophilic core-binding peptide comprises at least a portion of ApoE or an ApoE mimetic.

[0018] In certain embodiments, the affinity tag does not contain unpaired electrons (e.g., the affinity tag cannot act as a spin label). In other embodiments, the affinity tag comprises a peptide tag selected from the group consisting of: AviTag, calmodulin-tag, polyglutamic acid tag, FLAG-tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Softag 1, Sotftag 3, Strep-tag, TC tag, V5 tag, Xpress tag, Isopeptag, and SpyTag. In certain embodiments, the affinity tag is a tag based on click chemistry. In additional embodiments, the capture molecule is selected from the group consisting of: antibodies, streptavidin, calmodulin, nickel chelates, and cobalt chelates. In further embodiments, the capture molecule is bound to a solid support. In additional embodiments, the solid support is selected from beads, affinity columns, glass slides, or other available solid supports.

[0019] In certain embodiments, the initial sample is a blood sample, a serum sample, a plasma sample, or other biological fluid (e.g., urine). In certain embodiments, the initial sample is from a mammal (e.g., a dog, cat, horse, pig, or other livestock). In certain embodiments, the initial sample is from a human (e.g., a human at risk for or suffering from cardiovascular disease). In certain embodiments, the initial sample lacks LDL particles.

[0020] In certain embodiments, at least 90% of all the proteins in the purified sample are HDL lipoproteins (e.g., at least 90% ... 94% ... 98% ... 99% ... or at least 99.9%). In some embodiments, less than 10% of all the proteins in the purified sample are non-HDL lipoproteins (e.g., less than 10% ... 5% ... 1% ... 0.2%). In certain embodiments, the non-HDL lipoproteins are primarily or entirely serum albumin. In other embodiments, the method produces a purified sample from the initial sample in 1 hour or less (e.g., 1 hour ... 45 minutes ... 37 minutes ... 30 minutes ... 21 minutes ... 15 minutes ... or 10 minutes).

[0021] In certain embodiments, the method further comprises analyzing the purified sample to determine at least one characteristic of the group of labeled HDL molecules. In certain embodiments, the at least one characteristic comprises the level of cholesterol present in the group of labeled HDL molecules. In other embodiments, the labeled HDL molecules comprise at least one native ApoA1 protein, and wherein the at least one characteristic comprises determining the oxidation state of the native ApoA1 protein. In certain embodiments, the oxidation state of the native ApoA1 protein is determined (e.g., at one of the following tyrosine amino acid residues in the native ApoA1 protein: 29, 166, 192, and 236). In further embodiments, the analysis is performed using a technique selected from the group consisting of mass spectrometry (MS), chromatography, LC-MS, plasma resonance, and an analysis comprising the use of polyvinylsulfonic acid (PVS) and polyethylene glycol-methyl ether (PEGME). In certain embodiments, native ApoA1 from isolated HDL molecules is quantified (e.g., by mass spectrometry).

[0022] In certain embodiments, the at least one characteristic of the group of labeled HDL molecules is a cardiovascular disease risk marker for a subject and is used for the diagnosis and / or treatment of cardiovascular disease in the subject. In a specific embodiment, the cardiovascular disease marker comprises the HDL-c level in the subject. In a further embodiment, the treatment comprises administering to the subject a cardiovascular-related therapeutic agent (e.g., statin, ACE inhibitor, aldosterone inhibitor, angiotensin II receptor blocker, beta-blocker, calcium channel blocker, cholesterol-lowering drug, digoxin, diuretic, potassium, magnesium, vasodilator or warfarin) or a recommendation for lifestyle changes.

[0023] In certain embodiments, provided herein are methods comprising subjecting a sample comprising substantially purified non-fragmented ApoAl protein to mass spectrometry such that a mass spectrometry report (eg, electronic report, paper report, etc.) is generated for the non-fragmented ApoAl protein.

[0024] In certain embodiments, the mass spectrometry is performed at a resolution of at least 5000 full width at half maximum (FWHM) (e.g., at least 5000 ... 6000 ... 10,000 ... 15,000 ... 25,000 ... 30,000 ... 35,000 or more). In some embodiments, at least a portion of the non-fragmented ApoA1 protein comprises at least one modified amino acid (e.g., at least one, two, three, four or more modified amino acids) associated with an increased risk of cardiovascular disease. In certain embodiments, the spectrum report comprises a spectrum for the portion of the non-fragmented ApoA1 protein comprising at least one modified amino acid. In further embodiments, the modified amino acid is selected from the group consisting of a modified tyrosine, a modified tryptophan, and a modified methionine. In other embodiments, the modified tyrosine is at a position within ApoA1 selected from the group consisting of: 29, 166, 192, and 236. In certain embodiments, the modified methionine is at a position within ApoA1 selected from the group consisting of: 86, 112, and 148. In certain embodiments, the sample is from a subject, and wherein the method further comprises at least one of the following acts: i) informing the subject or the subject's physician that the subject is at increased risk for cardiovascular disease (CVD); ii) providing the subject or the subject's physician with a mass spectrometry report; iii) recommending, prescribing, or administering a CVD-related therapeutic agent to the subject; and iv) recommending, prescribing, or administering to the subject a follow-up test related to detecting CVD risk.

[0025] In certain embodiments, provided herein are methods comprising: a) subjecting a purified HDL sample to chromatography, such that a purified ApoA1 sample is produced that is substantially free of HDL-associated phospholipids, wherein the purified HDL sample comprises HDL molecules, and wherein the purified ApoA1 sample comprises non-fragmented ApoA1 protein; and b) subjecting the purified ApoA1 sample to mass spectrometry, such that a mass spectrometric report is produced regarding the non-fragmented ApoA1 protein.

[0026] In a further embodiment, the purified HDL is produced using the methods described herein (e.g., using an HDL marker molecule). In a further embodiment, the HDL molecule comprises: i) the non-fragmented ApoA1 protein, and ii) an HDL marker molecule, wherein the HDL marker molecule comprises: A) an HDL lipophilic core binding peptide, and B) an affinity tag. In a further embodiment, the processing in step a) and the processing in step b) are achieved by injecting the purified HDL sample into a device that performs chromatography and mass spectrometry. In some embodiments, the device is a liquid chromatography-mass spectrometry (LC / MS) machine. In additional embodiments, the mass spectrometry is performed at a resolution of at least 5000 full width at half maximum (FWHM).

[0027] In additional embodiments, at least a portion of the non-fragmented ApoA1 protein comprises at least one modified amino acid associated with an increased risk of cardiovascular disease. In other embodiments, the maximum spectrum report comprises a spectrum for a portion of the non-fragmented ApoA1 protein comprising at least one modified amino acid. In other embodiments, the modified amino acid is selected from the group consisting of a modified tyrosine, a modified tryptophan, and a modified methionine. In additional embodiments, the modified tyrosine is at a position within ApoA1 selected from the group consisting of: 29, 166, 192, and 236. In further embodiments, the modified methionine is at a position within ApoA1 selected from the group consisting of: 86, 112, and 148. In other embodiments, the sample is from a subject, and wherein the method further comprises at least one of the following acts: i) informing the subject or the subject's physician that the subject is at increased risk for cardiovascular disease (CVD); ii) providing a mass spectrometry report to the subject or the subject's physician; iii) recommending, prescribing, or administering a CVD-related therapeutic agent to the subject; and iv) recommending, prescribing, or administering to the subject a follow-up test related to detecting CVD risk.

[0028] In some embodiments, a system comprises: a) a device comprising a mass spectrometer; and b) a purified HDL sample comprising HDL molecules, wherein the HDL molecules comprise: i) non-fragmented ApoA1 protein, and ii) HDL marker molecules, each of the HDL marker molecules comprising: i) an HDL lipophilic core-binding peptide, and ii) an affinity tag.

[0029] In certain embodiments, provided herein are methods comprising: a) mixing an initial sample containing a population of HDL molecules and non-HDL biomolecules with a population of detectably labeled ApoA1 molecules to produce a mixed sample, wherein each of the HDL molecules comprises: i) an HDL lipophilic core and ii) a plurality of native ApoA1 proteins, and wherein the detectably labeled ApoA1 molecules are selected from the group consisting of: ApoA1 proteins, ApoA1 protein fragments, ApoA1 protein variants, and ApoA1 mimetics; b) incubating the mixed sample such that at least some of the ApoA1 molecules bind to at least some of the HDL molecules, thereby producing a population of labeled HDL molecules; c) purifying at least a portion of the population of labeled HDL molecules from the non-HDL biomolecules to produce a purified sample comprising the labeled HDL molecules; and d) analyzing the purified sample to determine a ratio of detectably labeled ApoA1 molecules to the native ApoA1 proteins. In certain embodiments, the ratio is used to determine the reverse cholesterol transport capacity of the HDL in the sample.

[0030] In certain embodiments, the detectably labeled ApoA1 molecule comprises a radiolabeled atom. In other embodiments, the detectably labeled ApoA1 molecule comprises a detectable label. In further embodiments, the detectable label is selected from the group consisting of a fluorescent label, an affinity tag, a chemiluminescent label, an antibody label, or an enzyme label. In further embodiments, analyzing the purified sample is performed using a method comprising mass spectrometry.

[0031] In certain embodiments, the amount of HDL captured via the affinity tag purification methods described herein is compared to the total amount of HDL in the initial sample in order to determine a ratio that serves as a proxy for the reverse cholesterol transport capacity of the HDL in the sample. Determination of total HDL can be performed by measuring HDL cholesterol, which is typically performed using a "homogeneous" assay that uses selected reagents added in a specific order to "clean" a serum sample of LDL cholesterol particles containing lipoprotein ApoB. Subsequently, HDL cholesterol is determined chemically using a conventional enzyme-coupled assay. Measuring total HDL can also be performed using physical methods of HDL particle separation, typically ultracentrifugation (e.g., Warnick et al., Clinical Chemistry, September 2001, Vol. 47, No. 9, 1579-1596, incorporated herein by reference).

[0032] In some embodiments, the amount of native ApoA1 captured via the affinity tag purification method described herein is compared to the total amount of native ApoA1 in the initial sample to determine a ratio that is used as a proxy for the reverse cholesterol transport capacity of HDL in the sample. ApoA1 is the major lipoprotein component of each HDL particle. Although the determination of HDL cholesterol rather than ApoA1 has been the primary support for cardiovascular risk assessment, this view is changing because the determination of ApoA1 can be used for the identification of subclinical atherosclerosis (Florvall et al., Journal of Gerontology: BIOLOGICAL SCIENCES 2006, Vol. 61A, No. 12, 1262-1266, incorporated herein by reference). Total ApoA1 is typically measured using a widely available immunoassay platform assay.

[0033] The present invention also includes the following items:

[0034] 1. A method for producing a purified sample, the method comprising:

[0035] a) mixing an initial sample containing a population of HDL molecules and non-HDL biomolecules with a population of HDL marker molecules to produce a mixed sample,

[0036] wherein each of the HDL molecules comprises: i) an HDL lipophilic core and ii) a plurality of HDL lipoproteins, and

[0037] wherein the HDL marker molecules each comprise: i) an HDL lipophilic core binding peptide, and ii) an affinity tag;

[0038] b) incubating the mixed sample such that at least some of the HDL marker molecules bind to at least some of the HDL molecules, thereby generating a population of labeled HDL molecules; and

[0039] c) purifying at least a portion of the population of labeled HDL molecules from the non-HDL biomolecules to produce a purified sample, wherein the purifying comprises contacting the mixed sample with a population of capture molecules specific for the affinity tag.

[0040] 2. The method of claim 1, wherein the HDL lipophilic core-binding peptide comprises the HDL binding region of apolipoprotein AI (ApoA1), and wherein the plurality of HDL lipoproteins comprises unlabeled ApoA1 molecules.

[0041] 3. The method according to item 2, wherein the HDL marker molecules are added to the initial sample such that the ratio of the HDL marker molecules to the unlabeled ApoA1 molecules is 1:2-2:1.

[0042] 4. The method of claim 2, wherein the plurality of HDL lipoproteins in each of the HDL molecules comprises a first and a second native ApoA1 protein, and wherein at least one of the HDL marker molecules displaces the first native ApoA1 protein in each of the HDL molecules when the labeled HDL molecules bind to the HDL molecules.

[0043] 5. The method of claim 2, wherein the HDL lipophilic core-binding peptide comprises at least a portion of ApoA1 or an ApoA1 mimetic.

[0044] 6. A method as described in claim 1, wherein the initial sample comprises a serum sample.

[0045] 7. The method of claim 6, wherein the serum sample is non-LDL-deficient.

[0046] 8. The method of claim 1 , wherein at least 90% of all of the proteins in the purified sample are the HDL lipoproteins.

[0047] 9. The method of claim 1 , wherein the method produces the purified sample from the initial sample in 1 hour or less.

[0048] 10. A composition comprising:

[0049] a) a group of HDL marker molecules, said molecules comprising:

[0050] i) at least a portion of ApoAl or an ApoAl mimetic that is capable of binding HDL, and

[0051] ii) an affinity tag; and

[0052] b) a population of unlabeled, wild-type ApoA1 molecules;

[0053] wherein the ratio of the HDL labeled molecules to the unlabeled molecules present in the composition is 1:2-2:1.

[0054] 11. The composition of claim 10, wherein the composition further comprises human serum.

[0055] 12. The composition of claim 11, wherein the human serum is non-LDL-deficient human serum.

[0056] 13. The composition of claim 10, wherein the affinity tag does not contain unpaired electrons.

[0057] 14. The composition of claim 10, wherein the unlabeled, wild-type ApoA1 molecule is part of an HDL molecule.

[0058] 15. A system comprising:

[0059] a) an HDL marker molecule, said molecule comprising:

[0060] i) HDL lipophilic core binding peptide, and

[0061] ii) an affinity tag; and

[0062] b) A population of capture molecules, wherein said capture molecules are specific for said affinity tag.

[0063] 16. The system of claim 15, wherein the HDL lipophilic core-binding peptide comprises the HDL binding region of apolipoprotein AI (ApoA1).

[0064] 17. The system of claim 15, wherein the HDL labeling molecule is in a first container, and wherein the population of capture molecules is in a second container.

[0065] 18. A composition comprising:

[0066] a) non-LDL-depleted serum samples; and

[0067] b) a group of HDL marker molecules, each comprising:

[0068] i) HDL lipophilic core binding peptide, and

[0069] ii) Affinity tag.

[0070] 19. The composition of claim 18, wherein the HDL lipophilic core-binding peptide comprises the HDL binding region of apolipoprotein AI (ApoA1), and wherein the non-LDL-depleted serum sample comprises unlabeled ApoA1 molecules.

[0071] 20. The composition of claim 19, wherein the HDL marker molecule is present in the non-LDL-depleted serum sample such that the ratio of the HDL marker molecule to the unlabeled ApoA1 molecule in the composition is 1:2-2:1.

[0072] Description of the drawings

[0073] Figure 1 The abundance of ApoA1 (the major HDL-associated protein) and serum albumin when isolated by the method in Example 1 is shown. Figure 1 B shows the abundance of ApoA1 and serum albumin when ApoA1 was purified from serum using ultracentrifugation.

[0074] Figure 2 Shown are SDS pages of various preparations from Example 1, including: 1) ladder; 2) serum (1:50 dilution); 3) Ni-NTA HDL preparation (10 ul); 4) UC HDL preparation (10 ul); and 5) ApoAl (purified from human, 5 ug).

[0075] Figure 3 An example mass spectrometry trace for intact ApoAl is shown. In this figure, charge states 32, 33, and 34 provide the most intense signals at nominal m / z values of 878, 851, and 826, respectively.

[0076] Figure 4A -C shows the complete detection results of ApoA1 and ApoA1 single oxidation. Specifically, Figure 4A Shown are the theoretical resolution of native and oxidized forms of ApoAl for the +35 charge state (+H adduct) using a mass spectrometer operating at a nominal resolution of 1000. Signal overlap between the two forms due to insufficient resolution is indicated. Figure 4B Shown are the theoretical resolution of native and oxidized forms of ApoAl for the +35 charge state (+H adduct) using a mass spectrometer operating at a nominal resolution of 2000. Signal overlap between the two forms due to insufficient resolution is indicated. Figure 4C The theoretical resolution of native and oxidized forms of ApoAl for the +35 charge state (+H adduct) is shown using a mass spectrometer operating at a nominal resolution of 10000. In this example, the peaks are well resolved from one another.

[0077] Figure 5A and 5B A low-resolution ion trap ( Figure 5A ) collected data for the +35 charge state of ApoA1 and the oxidized form of ApoA1, while the bottom graph ( Figure 5B) shows the same sample collected with the qTOF instrument operating at a nominal resolution of >30,000 FWHM.

[0078] Figure 6 Shows mass spectrometry data from a mixture of HDL proteins, how specific signals for ApoA1 and serum albumin can be selectively extracted by filtering specific signals. Top panel ( Figure 6 A) shows the total signal observed on the mass spectrometer during the chromatographic operation. Figure 6 B) shows the ApoA1 signal derived by filtering the data for the +35 charge state at m / z 803.38. Bottom panel ( Figure 6 C) shows contaminant serum albumin derived from the +54 charge state at m / z 1231.

[0079] Figure 7 Shown are bar graphs showing the recovery of labeled ApoAl and native HDL-associated proteins in neat serum with / without LDL depletion.

[0080] Figure 8 Shown are bar graphs showing the recovery of labeled ApoAl and native HDL-associated proteins from serum-purified HDL using increasing ratios of labeled ApoAl to native ApoAl.

[0081] Figure 9A and 9B Shown are A) amplification graphs showing RT-PCR of miRNA-223 and miRNA-16 (endogenous control) in rapidly purified HDL of two patient samples and a positive control, and B) bar graphs showing the relative abundance of amplified miRNA-223.

[0082] Figure 10A and 10B Shown are particle profiling analyses of human serum (A) and rapidly purified HDL (B) from the same sample.

[0083] definition

[0084] As used herein, "high-density lipoprotein" or "HDL" is a circulating, non-covalent assembly of amphiphilic proteins that enables the transport of lipids such as cholesterol and triglycerides in the water-based bloodstream. HDL is composed of approximately 50% by mass of amphiphilic proteins that stabilize a lipid emulsion consisting of a phospholipid monolayer (approximately 25%) embedded with free cholesterol (approximately 4%) and a core with triglycerides (approximately 3%) and cholesterol esters (approximately 12%). Subclasses of HDL include HDL2 and HDL3. HDL2 particles are larger and contain a higher content of lipids, while HDL3 particles are smaller and contain less lipids. Further subclasses include (from largest to smallest particles) HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c.

[0085] As used herein, "lipoprotein" refers to a type of protein to which one or more lipid molecules are attached or capable of being attached. In some cases, a lipoprotein may be a "lipid-poor lipoprotein," in which four or fewer molecules of phospholipids are bound. As used herein, lipoproteins include proteins that are not attached to lipids but can be exchanged in HDL particles (e.g., apolipoproteins).

[0086] As used herein, "sample" refers to a portion of a larger whole to be tested. Samples include, but are not limited to, body fluids such as blood, cerebrospinal fluid, urine, saliva, and the like.

[0087] As used herein, "blood sample" refers to a whole blood sample or the plasma or serum fraction thereof. In certain embodiments, a blood sample refers to a human blood sample, such as whole blood or the plasma or serum fraction thereof. In some embodiments, a blood sample refers to a non-human mammal ("animal") blood sample, such as whole blood or the plasma or serum fraction thereof.

[0088] As used herein, the term "whole blood" refers to a blood sample that has not been fractionated and contains cellular and fluid components.

[0089] As used herein, "plasma" refers to the fluid, non-cellular component of whole blood. Depending on the separation method used, plasma may be completely free of cellular components or may contain varying amounts of platelets and / or small amounts of other cellular components. Because plasma includes various coagulation factors, such as fibrinogen, the term "plasma" is distinguished from "serum," as described below.

[0090] As used herein, the term "serum" refers to whole mammalian serum, such as whole human serum, whole serum from test animals, whole serum from pets, whole serum from livestock, etc. In addition, as used herein, "serum" refers to plasma from which coagulation factors (e.g., fibrinogen) have been removed.

[0091] Details

[0092] The present invention provides methods, kits, and compositions for purifying HDL molecules from a sample (e.g., a blood sample) using an HDL labeling molecule comprising an HDL lipophilic core-binding peptide (e.g., a portion of ApoA1) and an affinity tag. In certain embodiments, the HDL purification is rapid (e.g., less than 1 hour) and allows determination of at least one cardiovascular risk factor (e.g., cholesterol level, oxidation state of ApoA1, etc.). The present invention also provides methods, kits, and compositions for detecting full-length ApoA1 without fragmenting ApoA1 using mass spectrometry. The present invention further provides methods, kits, and compositions for labeling HDL molecules in a sample with a detectably labeled ApoA1 molecule, such that the ratio of the detectably labeled ApoA1 molecule to the native ApoA1 protein can be determined.

[0093] I. HDL marker molecules

[0094] In certain embodiments, the present invention employs HDL marker molecules to add affinity tags to HDL molecules. Each HDL marker molecule comprises: i) an HDL lipophilic core-binding peptide, and ii) an affinity tag.

[0095] A. HDL lipophilic core binding peptide

[0096] The HDL lipophilic core binding peptide component of the HDL marker molecule can be any type of molecule that can bind to an HDL molecule (e.g., a mature HDL molecule) and can be linked to an affinity tag. The binding peptide can include, for example, at least the lipid binding portion of ApoA-I, ApoA-II, and ApoE.

[0097] ApoA-I is a lipoprotein that is a major component of HDL. An example of an apoA-I protein is human apoA-I protein (e.g., accession number NM_000039.1). Other examples of human apoA-I proteins are apoA-1 Milan and apoA-Iowa. The term also encompasses apoA-I proteins from non-human mammals, such as mouse, rat, rabbit, dog, pig, non-human primates, and the like. The term apoA-I also encompasses homologs of apoA-I. In certain embodiments, the HDL core-binding peptide comprises the lipid-binding portion of ApoA1.

[0098] ApoA-II is a lipoprotein that is the second most abundant component of HDL. An example of an ApoA-II protein is human ApoA-II protein (e.g., NP_001634). The term also encompasses ApoA-II proteins from non-human mammals, such as mice, rats, rabbits, dogs, pigs, non-human primates, and the like. In certain embodiments, the HDL-binding peptide comprises a lipid-binding portion of ApoAII.

[0099] ApoE refers to a lipoprotein involved in lipid metabolism and cholesterol transport. An example of an apoE protein is human apoE protein (e.g., NM_000041.2). There are three subtypes of human apoE protein: ApoE2, ApoE3, and ApoE4. ApoE3 is the major form of apoE, while apoE2 and apoE4 are distributed differently in lipoprotein particles (HDL, LDL, VLDL). The term also encompasses apoE proteins from non-human mammals such as mice, rats, rabbits, dogs, pigs, non-human primates, and the like. In certain embodiments, the HDL-binding peptide comprises a lipid-binding portion of ApoE.

[0100] In certain embodiments, ApoA1 proteins, fragments, or mimetics are used in HDL lipid-binding peptides, particularly portions of ApoA1 that are capable of binding to HDL. HDL-binding portions of ApoA1 are discussed, for example, in Murphy (ISRN Physiology, 2013, Article ID 186365; incorporated herein by reference). ApoA1 may include the full-length human ApoA1 peptide or a fragment or domain thereof (e.g., comprising a class A amphipathic helix). In certain embodiments, the HDL-binding peptide comprises an ApoA1 mimetic or fragment thereof. ApoA1 mimetics include, for example, naturally occurring variants of ApoA1 known in the art. For example, Weisgraber et al. have shown that cysteine can replace arginine at position 173 in a mutant form of ApoA1 known as ApoA1-milano (Weisgraber et al. (1983) J. Biol. Chem. 258:2508-2513, incorporated herein by reference). ApoAl polypeptide mimetics can also include polypeptides from ApoAl forms and variants, including, for example, apolipoprotein A-1 (Brewer et al., (1978)), apolipoprotein A-1 Milan (Weisgraber (1983)), apolipoprotein A-1 Paris (Bielicki and Oda (2002) Biochemistry 41:2089-2096), pre-apolipoprotein A-1, or any other mutant form of ApoAl known in the art, whether synthetically produced or naturally occurring.

[0101] In certain embodiments, the HDL-binding region of ApoA1 comprises amino acids 1-43 of SEQ ID NO: 1, or amino acids 5-38 of SEQ ID NO: 1, or amino acids 1-43 of SEQ ID NO: 1, except that one or two amino acids are deleted or altered without destroying the HDL-binding ability of the sequence. In other embodiments, the HDL-binding region of ApoA1 comprises amino acids 220-241 or 210-241 of SEQ ID NO: 1, or 223-238 or 220-241 of SEQ ID NO: 1, except that one or two amino acids are deleted or altered without destroying the HDL-binding ability of the sequence. In certain embodiments, the HDL-binding region of ApoA1 comprises amino acids 44-65 of SEQ ID NO: 1, or amino acids 47-62 of SEQ ID NO: 1, or amino acids 44-65 of SEQ ID NO: 1, except that one or two amino acids are deleted or altered without destroying the HDL-binding ability of the sequence. In certain embodiments, the HDL-binding region of ApoA1 comprises amino acids 1-43 and 220-241 of SEQ ID NO: 1, or amino acids 5-38 and 223-238 of SEQ ID NO: 1, or amino acids 1-43 and 220-241 of SEQ ID NO: 1, except that one or two amino acids are deleted or altered without destroying the HDL-binding ability of the sequence. In specific embodiments, the HDL-binding region of ApoA1 comprises amino acids 1-43 and / or 220-241 and / or 44-65 of SEQ ID NO: 1, or amino acids 5-38 and / or 223-238 and / or 47-62 of SEQ ID NO: 1, or said amino acid sequences, except that one or two amino acids are deleted or altered without destroying the HDL-binding ability of the sequence. Various HDL binding regions of human ApoA1 (SEQ ID NO: 1) are described in Frank and Marcel, 2000, J. Lipid Res., 41:853-872 and Tanaka, J. Pept. Sci., 2009, 15(1):36-42, both of which are incorporated herein by reference, particularly with respect to the sequences of ApoA1 and its HDL binding region. Figure 1Also specifically incorporated by reference. This figure shows the sequences of ApoA1 from baboon, dog, pig, rabbit, cow, hedgehog, mouse, rat, chicken, duck, and salmon. This figure allows the determination of the HDL-binding regions 1-43, 220-241, and 44-65 of the human sequence in these species. These sequences are expected to be the HDL-binding regions of ApoA1 in certain embodiments described herein. One skilled in the art can use the methods described by Frank and Marcel, Tanaka et al., and the following examples to determine whether a particular sequence of ApoA1 (e.g., with one or more amino acid changes) binds to HDL (e.g., by re-running the experiment with a candidate HDL-binding sequence).

[0102] Amino acid changes can be made to ApoA1, ApoA2, and ApoE, or fragments thereof, without disrupting their ability to bind to HDL lipoproteins. Such variants can be identified by analyzing the proposed variants using assays such as those described in the Examples below and testing them for HDL binding. Amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are all similar sizes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine are defined herein as biologically functional equivalents. Genes encoding for microbial expression of polypeptides having a primary conformation (e.g., substitutions, terminal and internal additions and deletions) that differ from the conformation specified herein with respect to the identity or position of one or more residues can be readily designed and manufactured according to the procedures described in the application published by Alton et al. (WO83 / 04053; incorporated herein by reference). Alternatively, modification of cDNA and genomic genes can be readily accomplished by the well-known technique of site-directed mutagenesis and used to produce analogs and derivatives of ApoAl, ApoAl, and ApoE.

[0103] B. Affinity Tag

[0104] The present invention is not limited by the affinity tag used as part of the HDL marker molecule. Examples of such tags include, but are not limited to, glutathione-S-transferase (GST), maltose binding protein (MBP), green fluorescent protein (GFP), AviTag (a peptide that allows biotin labeling by the enzyme BirA and thus the protein can be isolated by streptavidin), calmodulin-tag (a peptide bound by the protein calmodulin), polyglutamic acid tag (a peptide that binds efficiently to anion exchange resins such as Mono-Q), FLAG-tag (a peptide recognized by an antibody), HA-tag (a peptide recognized by an antibody), His-tag (typically 5-10 histidines bound by nickel or cobalt chelates), Myc-tag (a short peptide recognized by an antibody), S-tag, SBP-tag (a peptide that binds streptavidin), Softag 1. Strep-tag (peptide that binds streptavidin or a modified streptavidin called streptactin), TC tag (tetracysteine tag recognized by FlAsH and ReAsH bisarsenic compounds), V5 tag, Xpress tag, Isopeptag (peptide that covalently binds to pilin-C protein), and SpyTag (peptide that covalently binds to SpyCatcher protein). In certain embodiments, the tag is based on click chemistry.

[0105] The affinity tag can be directly coupled to the HDL phospholipid core-binding peptide, or can be separated by an intervening molecule such as a linker. In certain embodiments, a linker is used between the HDL lipophilic core-binding peptide and the affinity tag. Examples of suitable linkers include, but are not limited to, PEG linkers, peptide linkers, alkyl or substituted alkyl linkers, and the like. In some embodiments, the affinity tag and the HDL lipophilic core-binding peptide are directly conjugated, tethered, fused, etc. (e.g., via a covalent bond). In other embodiments, the two moieties are connected via a suitable linker. The present invention is not limited to any particular linker moiety. In some embodiments, the linker connects the two moieties. In some embodiments, the linker moiety covalently connects the two moieties. In some embodiments, the linker moiety is cleavable (e.g., chemically cleavable, enzymatically cleavable, etc.), such that exposure to appropriate conditions (e.g., a cleaving enzyme) cleaves the linker moiety and separates the connected moieties. In some embodiments, the linker moiety is a covalent bond that is: linear, branched, cyclic, heterocyclic, saturated, unsaturated, or a combination thereof. In some embodiments, the linker comprises 1-100 non-hydrogen atoms (plus hydrogen atoms) (e.g., 1-75, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, etc.) selected from the group consisting of C, N, P, O, and S. In some embodiments, the linker comprises any combination of alkyl, ether, thioether, polyether, amine, alkyl, amide, ester, carboxamide, sulfonamide, hydrazide linkages, and aromatic or heteroaromatic linkages. In some embodiments, the linker comprises a polymer (e.g., a nucleic acid, a polypeptide, a lipid, or a polysaccharide), a peptide linker, a modified peptide linker, a poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, a polyamino acid (e.g., polylysine), a functionalized PEG, a polysaccharide, a glycosaminoglycan, a dendrimer as described in WO 93 / 06868 and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29: 138-175 (1990), a PEG-chelator polymer as described in W94 / 08629, WO 94 / 09056, and WO 96 / 26754, an oligonucleotide linker, a phospholipid derivative, an alkenyl chain, an alkynyl chain, a disulfide, or a suitable combination thereof. In some embodiments, the linker moiety comprises any covalent or non-covalent molecular linker capable of stably stringing the first and second moieties together.

[0106] II. Detection Technology

[0107] The present invention is not limited by the methods used to detect HDL and / or ApoAl (eg, isolated using the methods described herein).

[0108] A. Detection Method

[0109] In certain embodiments, HDL (and associated ApoAl) isolated via the purification methods described herein is detected using a detection method selected from the group consisting of surface plasmon resonance, in vitro assays, activity assays, co-immunoprecipitation assays, mass spectrometry, fluorescence energy transfer (FRET), bioluminescence energy transfer (BRET), interferometry, biolayer interferometry (BLI), dual polarization interferometry ("DPI"), ellipsometry, and quartz crystal microbalance (see, e.g., U.S. Patent Publication No. 20130017556, herein incorporated by reference in its entirety).

[0110] B. Mass Spectrometry Detection of Intact ApoA1

[0111] In certain embodiments, methods are provided herein for detecting intact ApoA1 protein (i.e., undigested, full-length ApoA1) via mass spectrometry. The wild-type protein ApoA1 is encoded by a specific amino acid sequence. This sequence represents a functional protein after removal of a 24-amino acid precursor sequence and is shown in the following SEQ ID NO: 1:

[0112] DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLR AELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ(SEQ ID NO:1)

[0113] The mass of ApoA1 is derived from the atomic composition of ApoA1 based on the sequence. 1241 H 1977 N 347 O 389 S3, which gives a nominal, average neutral mass of 28078.26 Da.

[0114] In an exemplary embodiment, intact ApoA1 in serum or plasma can be detected by mass spectrometry by the following method. In preparation for separation and detection by LC / MS, intact ApoA1 protein is injected onto an HPLC column under substantially aqueous conditions (e.g., 94.8% water, 5% organic matter, and 0.2% acid, wherein the organic matter is typically methanol, acetonitrile, or isopropanol, and the acid is typically acetic acid or formic acid). Due to the hydrophobic nature of the protein, the ApoA1 protein binds to the column and salts and other hydrophilic contaminants are washed away under a constant flow of solvent. In order to distinguish ApoA1 from other proteins that may be present in the sample, the composition of the solvent flow through the column is adjusted to increase the percentage of organic modifier. This change can be adjusted in the sample or in a complex linear gradient or a series of steps so that proteins with different binding affinities can be eluted from the column under different solvent compositions. The eluate from the HPLC column can be transferred to multiple detectors (UV / Vis, light scattering, etc.). For detection by LC / MS, the eluent is sent to a mass spectrometer that detects molecules based on the behavior of gas phase particles so that they can be distinguished by their mass to charge (m / z) ratio. The first step in this process is to generate gas phase protein ions, which are typically generated by electrospray ionization. In this process, the solvent is removed from the protein molecules under conditions that allow hydrogen ions to remain adducted to the protein to form charged phase ions. In the electric field, the ions are attracted to the mass spectrometer where they are distinguished by their m / z ratio. In the case of multiple molecules, z can have a value greater than 1 and the full scan spectrum of ApoA1 is instructive. The spectrum is complex, with each peak in the spectrum corresponding to an ApoA1 with a specified charge state (z) for the signal. An exemplary spectrum of complete ApoA1 is shown in Figure 1. Figure 3 Shown in.

[0115] In principle, any of the identified charge states can be used to quantify ApoAl with distinct benefits / limitations. Figure 3In the chromatographic analysis, charge states 32, 33, and 34, at nominal m / z values of 878, 851, and 826, respectively, provide the strongest signals for selective detection. However, in certain embodiments, these strongest signals may not always be used if there are other co-eluting molecules that interfere with those ions. The charge state distribution for multiply charged ions can be modified depending on a variety of parameters, including mobile phase composition, heat and gas flow, and electric field strength. In addition, adducts other than hydrogen can also be used. For example, a sodium atom has a single positive charge but a mass of 23 Da. If ApoA1 at charge state 32 contained an adduct of 1 sodium and 31 protons, the nominal mass would be m / z 879. Therefore, adding other ionic species to the chromatographic solvent can, in certain embodiments, be a useful way to modify the charge state distribution. Adducts that can be used include, but are not limited to, sodium, potassium, lithium, and ammonium.

[0116] Mass spectrometry detection of intact ApoA1 can be used to identify modified (e.g., oxidized) forms of ApoA1. In certain embodiments, the modifications are relevant to cardiovascular disease detection and risk assessment. Detectable modifications include modified methionine (e.g., susceptible to sulfone formation), tryptophan oxidation, and tyrosine modifications (e.g., tyrosine chlorination, nitration, or bromination). The most relevant positions in ApoA1 for detecting cardiovascular disease risk with respect to tyrosine are positions 29, 166, 192, and 236 (see, e.g., U.S. Patent No. 8,338,110, incorporated herein by reference in its entirety). With respect to methionine, three positions are known to be particularly relevant (Met86, Met112, and Met148), all of which can be oxidized, converting the methionine to its sulfoxide form (see Pankhurst et al., J. Lipid Res., 44:349-355, 2003; Shao et al., J Lipid Res. 2010 Jul;51(7):1849-1858; and Shao et al., Chem Res Toxicol. 2010 Mar 15;23(3):447-454; each of which is incorporated herein by reference). In biological samples, a consequence of this process is that a population of ApoA1 molecules can exist in which the number of sulfoxides can range from 0 to 3. In situations where it is desirable to specifically determine the amount of ApoA1 and the specific contribution of each oxidized form in the population, the mass spectrometer should be capable of operating at a resolution sufficient to distinguish each form from the other. In Figures 4a-c, the effect on the resolution of the mass spectrometer is demonstrated. Using ApoA1 and ApoA1 with a single oxidation in a +35 charge state (m / z 803.38 and 803.84, respectively), modeled data from instruments with resolutions of 1000, 2000, and 10,000 FWHM are provided. At higher resolutions, the isotopic contribution due to overlapping lower charge oxidation states to higher charge states is minimized. In order to achieve a contribution due to isotopic overlap of less than 2%, the mass spectrometer should be operated at a resolution of 5000 FWHM or greater. The use of lower resolution instruments will generally necessitate the use of peak deconvolution to estimate and subsequently correct for overlapping signals. In certain embodiments, a high resolution mass analyzer such as a TOF or Orbitrap is employed and the analyzer prefers to use a low resolution ion trap or quadrupole. FIG5 shows a low resolution ion trap (top panel, Figure 5A ) collected on ApoA1 and ApoA1 oxidized form +35 charge state data. Bottom panel ( Figure 5B ) shows the same sample collected with the qTOF instrument operating at a nominal resolution of >30,000 FWHM.

[0117] Because mass spectrometry can resolve ions by mass, complex protein mixtures eluting in a mass spectrometer can be resolved if the resolution and mass differences are sufficient. Generating a chromatogram specific for a selected mass (extracted ion chromatogram – EIC) can generate a chromatogram specific for that molecule. Figure 6 Shows mass spectrometry data from a mixture of HDL proteins, how specific signals for ApoA1 and serum albumin can be selectively extracted by filtering specific signals. Top panel ( Figure 6 A) shows the total signal observed on the mass spectrometer during the chromatographic operation. Figure 6 B) shows the ApoA1 signal derived by filtering the data for the +35 charge state at m / z 803.38. Bottom panel ( Figure 6 C) shows contaminant serum albumin derived from the +54 charge state at m / z 1231.

[0118] III. Relationship between HLD, ApoA1, and Cardiovascular Disease

[0119] In certain embodiments, cardiovascular disease (CVD) or risk of CVD in a patient is detected by testing a patient sample using the methods described, employing mass spectrometric detection of intact ApoAl (eg, modified ApoAl) and / or the HDL purification protocols described herein.

[0120] For example, in certain embodiments, the method can be used to determine the ability of HDL to support reverse cholesterol transport. Reverse cholesterol transport (RCT) is a pathway for removing excess cholesterol from extrahepatic cells and tissues and ultimately transporting it to the liver for excretion, thereby reducing the accumulation of cholesterol in the arteries. The assessment of RCT is valuable, for example, for estimating overall cardiovascular risk and assessing the efficiency of possible therapies intended to accelerate RCT. Although the present invention is not limited to any particular mechanism, it is believed that the extent of ApoA1 exchange (for example, when adding labeled or otherwise labeled ApoA1 to a patient sample containing HDL) is directly related to its lipid efflux and carrying capacity. Therefore, in certain embodiments, free ApoA1 (for example, affinity-labeled ApoA1) is added to the system and analysis is used to determine how much of the added ApoA1 ultimately associates with the HDL particles.

[0121] An exemplary embodiment for performing the assessment is as follows. First, serum containing HDL is mixed with labeled ApoA1 so that endogenous ApoA1 can be identified from labeled ApoA1. The label may be incorporated, for example, via isotope incorporation, addition of a unique affinity tag, addition of additional amino acids, or chemical modification of the ApoA1 to be added. After the mixture equilibrates, it is expected that a certain proportion of HDL now contains labeled ApoA1. In certain embodiments, it may be necessary to remove excess labeled ApoA1 to facilitate measurement of the level of incorporation. Therefore, ultracentrifugation or other separation techniques that can distinguish HDL from unincorporated ApoA1 are employed. Finally, HDL is measured (if performed) using any suitable technique to determine the ratio of labeled ApoA1 to unlabeled ApoA1. In the method, high levels of ApoA1 incorporation indicate that the HDL molecule has a high level of reverse transport capacity (generally good for cardiovascular health), and HDL molecules with low levels of reverse transport capacity show an increased risk of cardiovascular disease.

[0122] A second exemplary embodiment is as follows. First, serum containing HDL is mixed with labeled ApoA1 so that endogenous ApoA1 can be distinguished from labeled ApoA1 and the label can be used to facilitate separation (e.g., an affinity tag is used as the label). After the mixture equilibrates, a certain proportion of the HDL will now contain labeled ApoA1. An affinity resin is then used to separate all labeled ApoA1 and any endogenous ApoA1 that has emerged through incorporation of the label into the HDL particles. Finally, the HDL is measured using any suitable technique to determine the ratio of labeled ApoA1 to unlabeled ApoA1. In this case, the amount of unlabeled ApoA1 is an important value because it is generated based on the degree of incorporation. It is also possible to determine the ratio of captured HDL to total available HDL.

[0123] In certain embodiments, the oxidation of ApoA1 is analyzed to assess CVD disease risk. Oxidized ApoA1 has a reduced cholesterol efflux stimulating activity as compared to unoxidized ApoA1. Therefore, detecting elevated levels of oxidized ApoA1 in patient samples using the compositions and methods described herein can be used to determine whether a subject is at risk for cardiovascular disease (see, e.g., U.S. Patent No. 8,338,110, incorporated herein by reference). In certain embodiments, tyrosine residues are interrogated, including positions 29, 166, 192, and 236 (e.g., to determine whether these positions are chlorinated or nitrated). Example

[0124] Example 1

[0125] Purification of HDL molecules from samples and characterization

[0126] This example describes methods for purifying HDL molecules using ApoAl molecules linked to an affinity tag, and methods for characterizing the purified HDL molecules.

[0127] Rapid separation of functional HDL

[0128] Human serum was depleted of LDL particles using conventional methods. Specifically, a 600 μL aliquot of human serum was mixed with 40 μL of a dextran sulfate / magnesium chloride solution. The sample was vigorously agitated, incubated at room temperature for 10 minutes, and the ApoB-containing precipitate was removed by centrifugation at 6,600 × g for 10 minutes. The supernatant was decanted and used for further experiments.

[0129] To achieve HDL purification, 12 μL of ApoB-deficient serum was mixed with 24 μL of affinity-tagged ApoA1 and 4 μL of PBS. The affinity tag in this example was polyhistidine. The sample was mixed vigorously and incubated at 37°C. After incubating the his-tagged ApoA1 with the ApoB-deficient serum, the sample was diluted with 500 μL of 10 mM imidazole buffer. While the present invention is not limited by any particular mechanism, and it should be understood that this mechanism is not necessary to practice the present invention, it is believed that his-tagged ApoA1 displaces one of the typically 4-7 native ApoA1 proteins on mature HDL molecules, thereby adding the tag to the mature HDL molecule. The sample was applied to a spin column containing Ni-NTA affinity media to capture the his-tagged ApoA1 and associated HDL. The spin column was briefly centrifuged to separate the his-tagged ApoA1 and associated HDL particles. The spin column was then washed with 500 μL of 20 mM imidazole buffer to remove non-specifically bound proteins. Finally, bound HDL particles were eluted by adding a 200 uL aliquot of 500 mM imidazole buffer.

[0130] Protein characterization

[0131] The purified HDL protein pool was analyzed by LC-MS and SDS-PAGE gel electrophoresis. For analytical separation prior to LC-MS, all forms of ApoA1 (native or labeled) were performed using a Waters column (50×0.75 μM, C18) using a heterogeneous, linear gradient of increasing concentrations of solvent B (acetonitrile + 0.2% formic acid) in solvent A (water + 0.2% formic acid). The HPLC eluate was directed to a Thermo Velos mass spectrometer operating in full scan mode.

[0132] Protein identification

[0133] HDL-associated proteins were determined using LC-MS / MS analysis of trypsin and Lys-c digests of isolated HDL particles. Three replicate preparations of the same serum sample, obtained using ultracentrifugation or affinity tag-purification, were digested at 37°C for 4 hours with the addition of the endoproteinase Lys-C. The resulting peptides were separated by nanoflow reversed-phase liquid chromatography (C18 column 75 μm id × 100 mm, 15 min. gradient) and detected by an LTQ-Orbitrap Elite mass spectrometer. The mass spectrometry data were searched using MaxQuant software, which uses an Android search engine to generate a list of proteins present in each sample.

[0134] Protein quantification

[0135] ApoAl was quantified using ELISA analysis.

[0136] PON1 activity

[0137] Pon1 is an HDL-associated protein with defined enzymatic activity. PON1 activity was determined by monitoring arylesterase activity using phenylacetate as a substrate according to Eckerson et al. (Am J Hum Genet. 1983 Nov; 35(6): 1126-1138).

[0138] Cholesterol efflux

[0139] Cholesterol efflux was assessed on a Vascular Strategies assay. The assay determines the ability of isolated HDL to transport cholesterol out of cells via the ABCA1 transporter.

[0140] result

[0141] The method allows for the rapid isolation of highly pure functional HDL particles from human serum / plasma under mild conditions.

[0142] Presence of HDL-associated proteins

[0143] A characteristic of HDL is the protein composition of the particle. Various studies have demonstrated that a variety of different proteins are associated with HDL, with ApoA1 being the major protein component (e.g., typically 4-7 ApoA1 proteins per HDL molecule). Although the mass spectrometry method used was not optimized for depth of proteome coverage, the list of identified protein IDs (Table 1 below) is in good agreement with the literature.

[0144] Table 1

[0145]

[0146] The highly enriched composition of HDL-associated proteins eluted from the affinity column demonstrates the successful isolation of HDL particles from serum using the affinity-tagged ApoA1 method described above. Only two nonspecific proteins (serum albumin and nucleobindin) were identified in the HDL preparation. Serum proteins are recognized as ubiquitous contaminants in all serum-based proteomics experiments. Nucleobindin is not reported as an HDL-associated protein and may represent a protein with nonspecific affinity for the nickel affinity resin used to capture his-tagged ApoA1.

[0147] Purity of rapidly isolated HDL particles

[0148] SDS page and LC-MS experiments confirmed the purity of the rapidly isolated HDL. Figure 1 A indicates the major HDL-associated protein ApoA1 from serum and its relative abundance when isolated by affinity methods. Figure 1 The purity of the affinity preparation is exemplary when compared to the high quality standard ultracentrifugation preparation shown in B. Figure 2 Analysis of intensity data from LC-MS and LC-MS / MS runs indicated that the his-tag purification contained approximately 12-fold less serum albumin than a comparable ultracentrifugation preparation.

[0149] Function of isolated HDL particles

[0150] HDL is known to have multiple biological functions, including lipid transport, cholesterol efflux, antioxidant and anti-inflammatory properties, and endothelial activation. Paraoxonase 1 is a bifunctional enzyme known to be associated with HDL, possessing both esterase and paraoxonase activities. Following rapid purification of HDL particles using affinity-tagged ApoA1, the isolated particles were shown to possess esterase activity. The particles were also shown to possess ABCA1-specific cholesterol efflux activity.

[0151] Exemplary Benefits of the ApoA1 Affinity Tag Purification Method

[0152] Two exemplary benefits of affinity ApoA1 purification by affinity chromatography are speed and purity. HDL preparation using affinity separation can be completed in 15 minutes. For example, a serum sample is mixed with an appropriate amount of affinity-tagged ApoA1 and incubated for 1-10 minutes to allow it to associate with HDL particles. After a brief equilibration (e.g., 1-2 minutes) with an affinity resin (NiNTA or Co-NTA beads), excess protein is washed off with buffer and eluted from the beads with a single application of imidazole or acid. This produces HDL with an apparent purity of >90% in 15 minutes or less.

[0153] In contrast, alternative methods for the isolation of HDL are generally more time-consuming. Equilibrium ultracentrifugation of HDL from human plasma generally takes 18-24 hours but produces high-quality HDL preparations that have been considered premium standards. Size exclusion chromatography can prepare one sample every two hours and has been widely used, but produces dilute fractions that are associated with generally lower purity, especially for smaller HDL-sized particles.

[0154] Example 2

[0155] Purification of HDL molecules from pure and LDL-depleted serum

[0156] This example describes the purification of HDL molecules using affinity-tagged ApoAl from LDL-depleted or pure (non-ApoB / LDL-depleted) serum.

[0157] Rapid separation of HDL

[0158] Human serum was depleted of LDL particles as described in Example 1 or used immediately for rapid HDL separation without LDL depletion. For rapid HDL purification, 12 uL of pure and LDL-depleted serum was mixed with 24 uL 15 N-labeled affinity-tagged ApoA1 was mixed. In this example, the affinity tag was polyhistidine. The sample was briefly mixed and incubated at 37°C. After incubation, the sample was diluted to 700uL with 10mM imidazole buffer. 25uL Ni-NTA affinity paramagnetic beads were added to the sample and briefly incubated to bind to the HDL molecules incorporated into the tagged ApoA1 and any additional unincorporated tags. The beads were washed twice with 300uL 20mM imidazole buffer in sequence to remove non-specifically bound proteins, followed by elution with 90uL 300mM imidazole buffer. 10uL 0.5ng / uL endoproteinase LysC was then added to the eluted HDL sample and incubated at 37°C for four hours to specifically cleave HDL-associated proteins into specific peptides for LC-MS characterization.

[0159] Purified HDL characterization

[0160] Peptide products from LysC digestion of rapidly purified HDL were separated on a Phenomenex reversed-phase HPLC column (3.0 × 50 mm, C18) using a heterogeneous, linear gradient of increasing concentrations of solvent B (acetonitrile + 0.1% formic acid) in solvent A (water + 0.1% formic acid). The eluted peptides were detected directly by an Agilent 6490 triple quadrupole mass spectrometer operated in multiple reaction monitoring mode to detect peptides specific for HDL-associated proteins.

[0161] result

[0162] Peptides specific for HDL-associated proteins as well as labeled ApoA-I were detected in pure serum and LDL-depleted serum samples. 15 N-enriched ApoA1 was used to distinguish the Figure 7 The intensities of labeled ApoAl and native HDL-specific ApoAl and ApoA2 are shown.These results indicate the ability to rapidly isolate HDL from patient serum without prior LDL depletion.

[0163] Example 3

[0164] Optimization of the labeled ApoA1:native ApoA1 ratio for rapid HDL purification

[0165] This example describes the rapid separation of HDL molecules in which the amount of labeled ApoAl is varied to maximize recovery of the molecule.

[0166] Rapid separation of HDL

[0167] For rapid HDL purification, 10 μL of pure (non-LDL-depleted) human serum was mixed with 24 μL of culture medium containing 1, 2, 5, 10, 20, 40, or 80 μg total labeled ApoA1 (corresponding to label:native ApoA1 ratios of 1:10, 1:5, 1:2, 1:1, 2:1, 4:1, and 8:1, respectively). 15 N-labeled affinity-tagged ApoA1 was mixed. The ratio was determined based on the assumption that the average total ApoA1 in a human serum sample was approximately 1 mg / mL (ug / uL). In this example, the affinity tag was polyhistidine. The sample was briefly mixed and incubated at 37°C. After incubation, the sample was diluted to 700uL with 10mM imidazole buffer. 25uL of Ni-NTA affinity paramagnetic beads were added to the sample and briefly incubated to bind to HDL molecules that had incorporated the tagged ApoA1, as well as any additional unincorporated tags. The beads were washed twice with 300uL of 20mM imidazole buffer to remove non-specifically bound proteins, followed by elution with 90uL of 300mM imidazole buffer. 10uL of 0.5ng / uL endoproteinase LysC was then added to the eluted HDL sample and incubated at 37°C for four hours to specifically cleave HDL-associated proteins into specific peptides for LC-MS characterization.

[0168] Purified HDL characterization

[0169] Peptide products from LysC digestion of rapidly purified HDL were separated on a Phenomenex reversed-phase HPLC column (3.0 × 50 mm, C18) using a heterogeneous, linear gradient of increasing concentrations of solvent B (acetonitrile + 0.1% formic acid) in solvent A (water + 0.1% formic acid). The eluted peptides were detected directly by an Agilent 6490 triple quadrupole mass spectrometer operated in multiple reaction monitoring mode to detect peptides specific for HDL-associated proteins.

[0170] result

[0171] Figure 8 The measured intensities of labeled ApoA1, native ApoA1, and native ApoA-II of purified HDL molecules from the same serum sample in which varying amounts of labeled ApoA1 were used to capture HDL are shown. 15 The labeled ApoA1 is distinguished from native ApoA-I by N isotope labeling, thereby producing a unique mass signature detectable by mass spectrometry. As expected, the signal intensity of the labeled ApoA1 increases when used at a larger label:native ratio. As illustrated in Example 1, although the present invention is not limited to any particular mechanism, and it should be understood that such a mechanism is not necessary to practice the present invention, it is believed that his-tagged ApoA1 replaces one of the typically 4-7 native ApoA1 proteins on the mature HDL molecule, thereby adding the tag to the mature HDL molecule. This is in Figure 8 The intensity of native ApoA1 in the HDL particle was observed to increase until a 1:1 ratio, then decrease as the tag:native ratio increased further. This is hypothesized to be a result of multiple ApoA1 molecules being displaced per HDL particle, thereby displacing native ApoA1 at a greater rate. Measurement of ApoA2, another HDL-specific protein that is not exchanged, serves as an indicator of total HDL recovery. ApoA2 was observed to increase to a maximum at a 1:1 ratio and to plateau as the tag:native ApoA1 ratio increased further.

[0172] Example 4

[0173] Characterization of ApoA1-labeled purified HDL

[0174] This example describes additional procedures for characterizing HDL isolated by the affinity-tagged methods described herein.

[0175] Fatty acid analysis

[0176] The ApoA-I (0.5 mg / mL) of His6 labeling is combined with human serum in a 1:2 volume ratio and incubated at 37°C for 15 minutes. The obtained sample is diluted to 700 μL with 10 mM imidazole, 50 mM sodium phosphate, 300 mM sodium chloride (pH 8.0) and incubated at room temperature for 10 minutes with paramagnetic beads containing Ni-NTA. The beads are washed twice with the serum extracted back and eluted with 30 μL 300 mM imidazole. The eluted HDL is combined with 500 μL 2% sulfuric acid in anhydrous methanol and heated at 65°C for 1.25 hours in a sealed vial. The obtained fatty acid methyl ester is extracted into 1 mL heptane using liquid-liquid extraction. The organic layer is removed and the heptane is evaporated under a stream of dry nitrogen. The fatty acid methyl ester is hydrolyzed into fatty acids by adding sodium hydroxide and subsequently analyzed by LC-MS for 19 common fatty acids.

[0177] The following fatty acids were detected in HDL in the following proportions: C14:0, myristic acid, 0.4%; C15:0, 0.1%, pentadecanoic acid; C16:0, 14.%, 1.6%, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid, 10.7%; C18:1, oleic acid, 19.7%; C18:2n6, 25.5%, linoleic acid; C18:3, linolenic acid, 1.2%; C20:0, arachidic acid; C20:1, trace%, eicosadienoic acid, 0.2%; C2 The following fatty acids were detected: C20:2n6, eicosadienoic acid, 0.2%; C20:3n6, homo-γ-linolenic acid, 4.2%; C20:4n6, arachidonic acid, 17.2%; C22:2n6, docosadienoic acid, 0.4%; C22:4n6, adrenic acid, 0.5%; C22:5n6, docosapentaenoic acid, 0.3%; C20:5n3, eicosapentaenoic acid, 0.8%; C22:6n3, docosahexaenoic acid, 1.9%; C22:5n3, docosapentaenoic acid, 0.6%. In the absence of labeled ApoA1, no fatty acids were detected. The composition of fatty acids detected in HDL samples differed from the whole blood fatty acid profile, showing an increased proportion of unsaturated fatty acids.

[0178] miRNA analysis

[0179] Total RNA from serum, rapid purified HDL (using the labeled ApoA1 method described herein) and positive serum control was isolated using the PureLink miRNA Isolation Kit (Life Technologies) and resuspended in nuclease-free water. Reverse transcription was performed using the TaqMan microRNA reverse transcription kit (Life Technologies). 5 μL total RNA (1-10 ng) was mixed with 1.0 mM dNTP, 3.33 U / μL reverse transcriptase, 1x reverse transcription buffer, 0.25 U / μL RNase inhibitor and a total of 12 ul of nuclease-free water. 3 μL 5x RT primers were then added to a total of 15 μL of RT reaction mixture. RT reactions were performed in an Eppendorf MasterCycler pro thermal cycler according to the manufacturer's instructions (30 minutes, 16°C; 30 minutes, 42°C; 5 minutes, 85°C; 4°C hold). cDNA was stored at -15°C to -25°C or used directly for quantitative analysis of miRNA.

[0180] TaqMan microRNA single analysis (analysis ID002295, Life Technologies) is used to evaluate mature miRNA-223 expression. Samples are standardized for miRNA-16 expression (analysis ID000391, Life Technologies). Regarding PCR reactions, 1.0ul TaqMan miRNA analysis and 1.33ul cDNA, 10ul TaqMan universal PCR reaction mixture II (without UNG) and 7.67ul nuclease-free water are mixed into the reaction mixture with a total of 20ul. All samples are operated in duplicate. Real-time PCR is performed in a Life Technologies Standard 7500 real-time PCR system, wherein the cycling conditions are 95°C for 10 minutes, followed by 45 cycles: 95°C for 15 seconds, then 60°C for 60 seconds. Comparative Ct analysis is performed to evaluate relative gene expression. The results shown in Figure 9 indicate differential miRNA-223 expression in different patient HDL samples, and its levels are similar to those of miRNA isolated from untreated serum samples (positive control).

[0181] Particle size analysis

[0182] His6-tagged ApoA-I (0.5 mg / mL) was combined with human serum in a 1:2 volume ratio and incubated at 37°C for 15 minutes. The resulting sample was diluted to 700 μL with 10 mM imidazole, 50 mM sodium phosphate, 300 mM sodium chloride (pH 8.0) and incubated with paramagnetic beads containing Ni-NTA for 10 minutes at room temperature. The beads were washed twice with back-extracted serum and eluted with 30 μL 300 mM imidazole in the back-extracted serum. 10 μL of HDL eluted in the back-extracted serum was separated by microfluidic electrophoresis using an Agilent 2100 Bioanalyzer. The resulting particle profile revealed that the peaks corresponding to the presence of HDL2, HDL2b, and HDL3 particles in the eluted sample ( FIG. 10 ) were at relative abundances similar to the same peaks in the unenriched serum sample.

[0183] All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions of the present invention will be readily apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in conjunction with specific preferred embodiments, it will be understood that the claimed invention should not be unduly limited to the specific embodiments. In fact, various modifications of the modes for carrying out the present invention that are readily apparent to those skilled in the relevant art are intended to be within the scope of the following claims. Sequence Listing <110> CLEVELAND HEARTLAB, INC. <120> Compositions and methods for the purification and detection of HDL and APOA1 <130> CHL-33754 / WO-1 / ORD <150> US 61 / 933,696 <151> 2014-05-15 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 243 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 1 Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala Thr 1 5 10 15 Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln 20 25 30 Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp 35 40 45 Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu 50 55 60 Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu 65 70 75 80 Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys 85 90 95 Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met 100 105 110 Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu 115 120 125 Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu 130 135 140 Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg 145 150 155 160 Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala 165 170 175 Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr 180 185 190 His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys 195 200 205 Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser 210 215 220 Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu 225 230 235 240 Asn Thr Gln

Claims

1. A method for producing a purified sample, the method comprising: include: (a) contacting a bodily fluid sample comprising high-density lipoprotein (HDL) molecules and non-HDL biomolecules with a group of HDL marker molecules to produce a mixed sample, the mixed sample comprising a group of labeled HDL molecules, wherein the HDL marker molecules each comprise: (i) an HDL lipophilic core-binding peptide comprising a lipid-binding portion of apolipoprotein (Apo) Al, wherein the lipid-binding portion of ApoAl comprises one or more of the following: amino acids 1-43 of SEQ ID NO: 1, amino acids 5-38 of SEQ ID NO: 1, amino acids 220-241 of SEQ ID NO: 1, amino acids 210-241 of SEQ ID NO: 1, amino acids 223-238 of SEQ ID NO: 1, amino acids 44-65 of SEQ ID NO: 1, and amino acids 47-62 of SEQ ID NO: 1; and (ii) an affinity tag, and wherein the group of labeled HDL molecules comprises at least some of the HDL marker molecules that bind to at least some of the HDL molecules; (b) purifying at least a portion of the group of labeled HDL molecules from the non-HDL biomolecules by contacting the mixed sample with a group of capture molecules specific for the affinity tag to produce a purified sample, wherein at least 90% of all proteins in the purified sample are said HDL molecules, and optionally wherein the purified sample is produced from the body fluid sample in 1 hour or less.

2. The method of claim 1, wherein the body fluid sample is a blood sample or a urine sample, optionally wherein the blood sample is a serum sample or a plasma sample, and optionally wherein the blood sample comprises low-density lipoprotein (LDL) molecules.

3. The method of claim 1 or 2, wherein each of the HDL molecules comprises apolipoprotein (Apo) AI, Apo-AII or ApoE, optionally wherein the body fluid sample comprises HDL2 HDL2b or HDL3 molecules.

4. The method of any one of claims 1-3, wherein generating the purified sample further comprises chromatography, optionally wherein the chromatography is affinity chromatography, and optionally wherein the method further comprises subjecting the purified sample to mass spectrometry.

5. The method according to any one of claims 1-4, further comprising depleting the body fluid sample of low-density lipoprotein (LDL) molecules before step (a), optionally wherein depleting the body fluid sample of LDL molecules comprises contacting the body fluid sample with a dextran sulfate / magnesium chloride solution and collecting the supernatant, and optionally wherein the supernatant is contacted with the HDL marker molecule in step (a).

6. A method for determining the reverse cholesterol transport capacity of high-density lipoprotein (HDL) molecules, wherein include: (a) contacting a bodily fluid sample comprising HDL molecules with a group of HDL marker molecules to produce a mixed sample comprising a group of labeled HDL molecules, wherein the HDL marker molecules each comprise: (i) an HDL lipophilic core-binding peptide comprising a lipid-binding portion of apolipoprotein (Apo) Al, wherein the lipid-binding portion of ApoAl comprises one or more of the following: amino acids 1-43 of SEQ ID NO: 1, amino acids 5-38 of SEQ ID NO: 1, amino acids 220-241 of SEQ ID NO: 1, amino acids 210-241 of SEQ ID NO: 1, amino acids 223-238 of SEQ ID NO: 1, amino acids 44-65 of SEQ ID NO: 1, and amino acids 47-62 of SEQ ID NO: 1; and ii) an affinity tag, and wherein the group of labeled HDL molecules comprises at least some of the HDL marker molecules that bind to at least some of the HDL molecules; and (b) measuring the amount of HDL to determine the ratio of labeled HDL molecules to non-labeled HDL molecules, wherein a high ratio of labeled HDL molecules to non-labeled HDL molecules indicates that the HDL molecules have a high level of reverse cholesterol transport ability, wherein the labeled HDL molecules are His-tagged apolipoprotein (Apo)A1, and the non-labeled HDL molecules are non-labeled ApoA1.

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