CPC G01N 33/92 (2013.01) [G01N 2333/775 (2013.01)] | 11 Claims |
1. A method of extracting and detecting two or more HDL-associated proteins comprising:
a) mixing, in a first container, a separating solution and a serum or plasma sample to generate a first mixed sample, wherein said separating solution comprises a strong organic acid and a hydrophilic organic solvent, and wherein said separating solution makes up 55%-93% of said first mixed sample;
b) exposing said first mixed sample to centrifugal force such that said first mixed sample separates into a pellet and supernatant;
c) transferring at least a portion of said supernatant to a second container such that it is separated from said pellet;
d) adding a non-polar organic solvent to said second container at a ratio of greater than 1:1 to generate a second mixed sample;
e) exposing said second mixed sample to centrifugal force such that said second mixed sample separates into an upper organic solvent layer and a bottom aqueous layer;
f) transferring at least a portion of said bottom aqueous layer to a third container such that it is separated from said upper organic solvent layer;
g) adding a protease to at least a portion of said bottom aqueous layer, wherein the protease is selected from trypsin, LysC, GluC, ArgC, AspN, chymotrypsin, and pepsin; and
h) subjecting at least a portion of said bottom aqueous layer to a detection assay such that at least two or more HDL-associated proteins are detected, wherein a first HDL-associated protein is Apolipoprotein A-I (APOAI) and a second HDL-associated protein is selected from Apolipoprotein A-II (ApoA2), Alpha-1-antitrypsin (SERPINA1), Apolipoprotein C-III (APOC3), Apolipoprotein C-I (APOCI), Apolipoprotein A-IV (APOA4), Apolipoprotein C-II (APOC2), Angiotensinogen (AGT), Complement C3 (C3), Complement C4-B (C4B), Serum amyloid A-4 protein (SAA4), Transthyretin (TTR), Pigment epithelium-derived factor (SERPINF1), Apolipoprotein L1 (APOL1), Apolipoprotein F (APOF), Apolipoprotein M (APOM), Apolipoprotein Cl (APOC1), Fibrinogen alpha chain (FGA), Apolipoprotein E (APOE), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Serum amyloid A-1 protein (SAA1), Alpha-2-HS-glycoprotein (AHSG), Complement C4-A (C4A), Retinol-binding protein 4 (RBP4), or Apolipoprotein D (APOD).
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