Abstract 548: Separation of Lipoprotein Particles Utilizing Asymmetric Flow Field-flow Fractionation and Quantitation of Apolipoprotein L1 in Human Plasma with IMER-UPLC-MS/MS

Apolipoprotein L1 (ApoL1) is an intriguing protein and was shown to be involved in numerous diseases states such as chronic kidney disease (CKD), cardiovascular diseases(CVD), Human Immunodeficiency Virus associated Nephropathy (HIVAN), systemic lupus erythematosus (SLE) (associated collapsing glomerulopathy), and type 2 diabetes(especially for people of African descent). People of African ancestry were shown to have either one or two alleles which contribute to their susceptibility of diseases related to CKD. ApoL1 is characterized as a HDL binding protein. In this work we studied ApoL1 binding to HDL by using a gentle size fractionation technique, asymmetric flow field-flow fractionation (AF4), and liquid chromatography (LC) with quantitative tandem mass spectrometry (MS/MS) detection. We injected 50 uL of whole plasma onto the AF4 system, where smaller HDL particles eluted first followed by the larger HDL, and then by LDL, IDL and VLDL particles. The lipoproteins were separated into 40 fractions and quantitatively analyzed using on-line trypsin digestion coupled with LC-MS/MS analysis. We analyzed plasma samples both with and without purification by UC. When HDL particles were separated by UC, apoL1 was found in the higher density HDL fractions, as was also found by previous studies. When HDL separated using AF4 with or without UC, apoL1 eluted in the larger HDL size region (12-13 nm). With UC vs. without UC, the apoL1 peptide signal intensities were reduced by more than 50%, indicating significant loss of apoL1 from the surface of HDL particles during UC. Our data on apoL1 provides further evidence for the fact that size and density are not directly interconvertible physical characteristics of lipoproteins. Furthermore, our data shows that apoL1 is also an example of exchangeable apolipoproteins whose binding can be significantly diminished due to the effect of intense shear forces during UC separation. This work also demonstrates the advantages of utilizing AF4-IMER-UPLC-MS/MS methodology to separate lipoprotein particles and study of their apolipoprotein composition.