Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modelling of atherosclerosis has been the mainstay for pre-clinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity and it is critical to monitor lipoprotein levels during pre-clinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during pre-clinical experiments limit analyses to measuring total cholesterol and -triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple-reaction-monitoring (MRM)-based mass spectrometry assay for simultaneous measurement of nine apolipoproteins using few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: The widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay with stable isotope dilution (SID) that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during pre-clinical investigations. 

Plasma pools from genotype and feeding according to Table S1. An equal plasma volume was pooled from each mouse within a treatment group. SEC analysis (Superose 6 Increase 10/300 GL) was carried out using a total of 0.5 mL plasma collected in fractions of 0.25 mL in a 96-well plate. A total of 36 fractions from each condition was subjected to sample prep for MRM analysis.

Table S1