Compositions and methods for purification and detection of HDL and ApoA1
The present invention provides methods, kits, and compositions for purifying HDL molecules from a sample (e.g., blood sample) using HDL tagging molecules comprising an HDL lipophilic core binding peptide (e.g., portion of ApoA1) and an affinity tag. The present invention also provides methods, kits, and compositions for detecting non-fragmented ApoA1 with mass spectrometry. The present invention further provides methods, kits, and compositions for tagging HDL molecules in a sample with detectably labeled ApoA1 molecules such that the ratio of detectably labeled ApoA1 molecules to native ApoA1 proteins may be determined.
1. A method of generating a purified sample comprising:
a) mixing an initial human serum, plasma, or whole blood sample containing a population of HDL molecules and non-HDL biomolecules with a population of HDL tagging molecules to generate a mixed sample,
wherein said HDL molecules each comprise: i) an HDL lipophilic core and ii) a plurality of HDL lipoproteins, and
wherein said HDL tagging molecules each comprise: i) an HDL lipophilic core binding peptide which comprises an HDL binding region of human Apolipoprotein A-I (ApoA1), and ii) an affinity tag;
b) incubating said mixed sample such that at least some of said HDL tagging molecules bind to at least some of said HDL molecules, thereby generating a population of tagged HDL molecules; and
c) purifying at least a portion of said population of tagged HDL molecules away from said non-HDL biomolecules to generate a purified sample,
wherein said purifying comprises contacting said mixed sample with a population of capture molecules that are specific for said affinity tag, and
wherein said tagged HDL molecules in said purified sample have esterase activity and ABCA1 specific cholesterol efflux activity.
2. The method of claim 1 , wherein said HDL tagging molecules are added to said initial sample such that the ratio of said HDL tagging molecules to said non-tagged ApoA1 molecules is 1:2-2:1.
3. The method of claim 1 , wherein said HDL lipophilic core binding peptide comprises ApoA1.
4. The method of claim 1 , wherein said initial sample comprises a serum sample.
5. The method of claim 4 , wherein said serum sample is non-LDL depleted.
6. The method of claim 1 , wherein at least 90% of all the proteins in said purified sample are said HDL lipoproteins.
7. The method of claim 1 , wherein said method generates said purified sample from said initial sample in 1 hour or less.